Beta secretase exosite binding peptides and methods for identifying beta secretase modulators

ABSTRACT

The present invention provides peptides that specifically bind to BACE at a newly discovered exosite. The invention also provides methods for identifying peptides that bind to a BACE exosite. The invention further provides methods for identifying compounds that bind to a BACE exosite and modulate BACE activity. In another aspect, the invention provides methods for treating or preventing neurodegenerative disorders such as Alzheimer&#39;s disease by administering compounds that bind to a BACE exosite and modulate BACE activity.

[0001] The present patent application claims the benefit of U.S.Provisional Patent Application Serial No. 60/418,679, filed Oct. 15,2002, the disclosure of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

[0002] The present invention relates to peptides that bind to betasecretase (“β-secretase”) at a newly discovered exosite within thecatalytic domain of the enzyme, and use of these peptides and variantsthereof to identify therapeutic molecules useful for the treatment ofneurological disorders.

BACKGROUND OF THE INVENTION

[0003] Alzheimer's disease (“AD”) is a devastating neurodegenerativedisease that affects millions of elderly patients worldwide. AD ischaracterized clinically by progressive loss of memory, orientation,cognitive function, judgement and emotional stability. With increasingage, the risk of developing AD increases exponentially, so that by age85 some 20-40% of the population is affected. Memory and cognitivefunction deteriorate rapidly within the first 5 years after diagnosis ofmild to moderate impairment, and death due to disease complications isan inevitable outcome. AD is the most common cause of nursing homeadmittance in the United States; hence, in addition to the morbidity andmortality experienced by the patient, there are considerable economicand emotional burdens placed on the family, caregivers and society atlarge. The only recognized treatment currently available for AD isacetylcholinesterase inhibitors, which merely treat the symptoms ofcognitive impairment. No method for prevention or treatment of thepathophysiology of AD is currently available.

[0004] Diagnosis of AD is based mainly on subjective assessments ofmemory and cognitive function. Definitive diagnosis can only be madepost-mortem, based on histopathological examination of brain tissue fromthe patient. Two histological hallmarks of AD are the occurrence ofneurofibrillar tangles of hyperphosphorylated tau protein and ofproteinaceous amyloid plaques, both within the cerebral cortex of ADpatients. The amyloid plaques are composed mainly of a peptide of 39 to42 amino acids designated beta-amyloid, also referred to as β-amyloid,amyloid beta, Aβ, βAP, β/A4; and referred to herein as beta-amyloid andAβ. It is now clear that the Aβ peptide is derived from a type 1integral membrane protein, termed beta amyloid precursor protein (alsoreferred to as “β-APP” and “APP”) through two sequential proteolyticevents. First, the APP is hydrolyzed at a site N-terminal of thetransmembrane alpha helix by a specific proteolytic enzyme referred toas β-secretase. The soluble N-terminal product of this cleavage eventdiffuses away from the membrane, leaving behind the membrane-associateC-terminal cleavage product, referred to as C99. The protein C99 is thenfurther hydrolyzed within the transmembrane alpha helix by a specificproteolytic enzyme referred to as γ-secretase. This second cleavageevent liberates the Aβ peptide and leaves a membrane-associated “stub”.The Aβ peptide thus generated is secreted from the cell into theextracellular matrix where it eventually forms the amyloid plaquesassociated with AD.

[0005] Several lines of evidence suggest that abnormal accumulation ofAβ plays a key role in the pathogenesis of AD. First, Aβ is the majorprotein component of amyloid plaques. Second, Aβ is neurotoxic and maybe causally linked to the neuronal death associated with AD. Third,missense DNA mutations at several positions within the APP protein canbe found in affected members but not unaffected members of severalfamilies with a genetically determined (familial) form of AD. Forexample, one familial form of AD is linked to a pair of mutations,referred to as the “Swedish mutations”, that are immediately proximal tothe site of β-secretase-mediated hydrolysis of APP (Mullan et al.,(1992) Nature Genet. 1:345-347). Patients bearing the Swedish mutantform of APP develop AD at a much earlier age (typically within thefourth decade of life) and likewise progress to severe dementia at amuch earlier age. Histopathological examination of the brains ofpatients suffering from the “Swedish mutant” form of familial AD isidentical to that of brains from patients suffering from non-familial,sporadic forms of the disease. It is therefore hypothesized that haltingthe production of Aβ will prevent and/or reduce the neurodegenerationand other pathologies of AD. One method of halting Aβ production wouldbe to administer specific inhibitors of one or both of the proteolyticenzymes involved in APP processing, namely, β-secretase and γ-secretase.The molecular identity of the protein responsible for γ-secretaseactivity has not yet been determined, although there is a preponderanceof data suggesting a role for the proteins presenilin-1 and presenilin-2in this enzymatic action. Nevertheless, compounds that inhibit theaction of γ-secretase, and thus inhibit Aβ production in cell culturehave been identified by several groups.

[0006] Recently the molecular identity of the protein responsible forβ-secretase activity has been determined and this protein is commonlyreferred to as BACE (for Beta-site APP Cleaving Enzyme). This enzyme isa type 1 membrane protein that folds into an extra-membranous globularcatalytic domain that is tethered to the membrane by a single alphahelix. The catalytic domain of BACE contains the canonical signaturemotifs for an aspartyl protease, and the enzymatic activity ofrecombinant versions of the catalytic domain of human BACE is consistentwith this designation. It is well known that aspartyl proteases can beeffectively inhibited by small molecules and peptides that bind to, andhence block, the site on the enzyme molecule at which the chemicaltransformations of the substrate molecule takes place. This site ofchemical reactivity is commonly referred to as the enzyme active site.For aspartyl proteases this site contains the two chemically reactiveaspartic acid residues from which this class of enzymes derive its name.During the course of enzymatic action on the substrate molecule, theenzyme goes through an intermediate state in which the carbonyl carbonof the hydrolyzable amide bond of the substrate forms four coordinatebonds, engaging the active site aspartic acid residues of the enzyme.

[0007] A common strategy for inhibiting aspartyl proteases is to preparea small peptide of amino acid composition similar to the substratemolecule but replacing the hydrolyzable amide bond with a chemical groupthat mimics the four coordinate carbon intermediate species justdescribed. It is well known that chemical groups such as statines,hydroxyethylenes, hydroxyethylamines and similar structures are veryeffective for this purpose. Indeed, peptidic inhibitors of BACE,incorporating statine and hydroxyethylene structures have been reported.Recently the 3-dimensional structure of the catalytic domain of humanBACE in complex with a hydroxyethylene-based peptidic inhibitor referredto as OM99-2 has been solved by the methods of x-ray crystallography.The resulting structure confirmed that the inhibitor binds within theenzyme active site, engaging the active site aspartic acid residues asexpected. Hence, active site-directed inhibitors of BACE can be designedand may prove useful as pharmacological agents for the treatment of AD.Historically, however, it has proved difficult to develop molecules ofpharmacological utility based on active site-directed inhibitors ofaspartyl proteases. While very potent inhibitors have been identified invitro, active site-directed inhibitors of aspartyl proteases may presentin vivo issues of oral bioavailability and pharmacokinetic half-life.

[0008] In addition to the active site, some proteolytic enzymes containadditional binding pockets that engage the substrate protein atlocations distal to the site of chemical transformation. These bindingpockets are referred to as exosites and can contribute significantly tothe stabilization of the enzyme-substrate binary complex by providingimportant structural determinants of interaction. Additionally, exositeson some proteolytic enzymes can act as allosteric regulators of enzymeactivity, so that binding interactions at the exosite are transmittedthrough conformational changes of the enzyme to the active site, wherestructural changes can augment or diminish the chemical reactivity ofthe active site. In some cases molecules have been identified that bindto an exosite, rather than the active site, of proteolytic enzymes andthese have proved to be effective inhibitors of enzymatic action. Hence,exosites represent an alternative target for inhibitory ligand bindingto proteolytic enzymes. Because the exosites are distinct from theactive sites of these enzymes, the nature of the molecules that bind tothe exosites can be very different from active site-directed inhibitors.In favorable cases, the nature of the molecules binding to the exositesare more pharmacologically tractable relative to the activesite-directed inhibitors of the same enzyme.

SUMMARY OF THE INVENTION

[0009] The present invention provides peptides that specifically bind toBACE at a newly discovered exosite within the catalytic domain of theenzyme, and are referred to herein as “exosite binding peptides” or“EBPs”. The peptides of the present invention can be used to modulateBACE activity and interfere with hydrolysis of APP and APP-derivedsubstrates.

[0010] The invention also provides methods for identifying peptides thatbind to a BACE exosite comprising contacting BACE with at least onepeptide, and determining whether the peptide specifically binds to BACEat a site other than the active site of BACE.

[0011] The invention further provides methods of using the peptides andvariants thereof for identifying compounds that bind to BACE exositesand modulate BACE activity. In another aspect, the invention providesmethods for treating or preventing neurological disorders such asAlzheimer's disease by administering compounds that bind to a BACEexosite and inhibit beta-amyloid production.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows isothermal calorimetry data quantitativelydetermining the binding affinity of peptide NLTTYPYFIPLP (SEQ ID NO:19)to BACE at 25° C., in Dulbecco's PBS wherein the parameters wereK_(A)=1.65×10⁷ M⁻¹; K_(d)=61 nM; n=1.03; and ΔH=−12.9 kcal/mol.

[0013]FIG. 2 shows isothermal calorimetry data quantitativelydetermining the binding affinity of peptide ALYPYFLPISAK (SEQ ID NO:20)to BACE at 25° C. in Dulbecco's PBS wherein the parameters wereK_(A)=8.86×10⁶ M⁻¹; K_(d)=113 nM; n=1.06; and ΔH=−11.8 kcal/mol.

[0014]FIG. 3 shows integrated isothermal calorimetry data quantitativelydetermining the binding affinities of BACE-OM99-2 titrated with peptideNLTTYPYFIPLP (SEQ ID NO:19) in pH 5.3 buffer at 37° C.

[0015]FIG. 4 shows EBPs labeled with the fluorescent group Alexa488 atdifferent positions (Molecules X, Yn and Z of Example 9) and withlinkers of different lengths (Yn, wherein n=1-4).

[0016]FIG. 5 shows fluorescence anisotropy data demonstrating thatlabeled EBP Molecule X binds to BACE at pH 7.1 (upper panel) and pH 4.5(lower panel). The solid lines represent fitting the data to a 1:1binding model.

[0017]FIG. 6 shows that BMS-561871, peptide NLTTYPYFIPLP (SEQ ID NO:19)(Molecule X without the Alexa488 label) competes with the binding ofMolecule X at pH 7.1 (upper panel) and pH 4.5 (lower panel), asmonitored by fluorescence anisotropy.

[0018]FIG. 7 shows inhibition of the binding of Molecule X to BACE by acollection of N-terminally (upper panel) and C-terminally (lower panel)truncated unlabeled peptides monitored by fluorescence anisotropy. Thenumbered identifiers on the X-axis refer to the amino acid compositionwith respect to BMS-561871.

[0019]FIG. 8 shows the binding of Molecule Y1 to BACE at pH 4.5 byfluorescence anisotropy. The solid line represents fitting the data to a1:1 binding model.

[0020]FIG. 9 shows that BMS-593925, peptide YPYFIPL (SEQ ID NO:10)(Molecule Y1 without the Alexa488 label) competes with the binding ofMolecule Y1 at pH 4.5 monitored by fluorescence anisotropy.

[0021]FIG. 10 shows the fluorescence anisotropy measurement of MoleculeY1 binding to BACE both in the absence (circles) and in the presence(squares) of OM99-2, an active site-directed inhibitor of BACE,suggesting that Molecule Y1 does not bind to BACE at the active site.

[0022]FIG. 11 shows the inhibition of the binding of Molecule Y1 to BACEby a collection of mutated peptides based on peptide YPYFIPL (SEQ IDNO:10) (wt sequence, corresponding to 5-11 in the original NLTTYPYFIPLPpeptide; SEQ ID NO:19): Upper panel: Peptides with a mutation to Ala atthe indicated position. Lower panel: Peptides with a mutation tobenzophenone (Bpa or B) at the indicated position. N—B and C—B representpeptides that have the Bpa group attached to the N- and C-terminus,respectively. Peptides P6B, I9B, and C—B (indicated by asterisks in thefigure) have limited solubility, therefore, the inhibition of bindingbetween Molecule B1 and BACE is under determined in these cases.

[0023]FIG. 12 shows a fluoroimage (excitation at 488 nm, emission at 530nm) of the time course of the photo-crosslinking of BACE (2 μM) to aBpa-containing EBP, BMS-607641, with the sequence of YPYFIPB-Alexa488 (2μM) (SEQ ID NO:108) in the presence of 100 μM of a scrambled peptide(BMS-599271, LYPPYIF; SEQ ID NO:53) at 4° C.

[0024]FIG. 13 shows that Molecule Y3 inhibits the proteolytic activityof BACE monitored by HPLC analysis. The solid line represents fitting ofthe data to the Langmuir isotherm equation (Copeland. R. A., Enzymes: APractical Introduction to Structure, Mechanism, and Data Analysis,(2^(nd) ed), Wiley-VCH, New York, N.Y. (2000)).

[0025]FIG. 14 shows integrated and fitted binding data for BACE andBMS-655507 (Ac-His-Trp-Pro-Phe-Phe-Ile-Arg-Ser; SEQ ID NO:57), at 25°C., in Dulbecco's PBS; parameters: K_(A)=1.09×10⁶ M⁻¹; K_(d)=0.914 μM;n=0.71; ΔH=−18.04 kcal/mol.

[0026]FIG. 15 shows integrated and fitted binding data for BACE andBMS-655507 (Ac-His-Trp-Pro-Phe-Phe-Ile-Arg-Ser; SEQ ID NO:57), at 25°C., in 50 mM NaOAc, pH 4.5; parameters: K_(A)=6.10×10⁵ M⁻¹; K_(d)=1.64μM; n=0.86 ; ΔH=−13.35 kcal/mol.

DETAILED DESCRIPTION OF THE INVENTION

[0027] In accordance with the present invention, we have discoveredpeptides that specifically bind to a Beta-site APP Cleaving Enzyme(BACE) binding site that is not the BACE active site. The term “BACEexosite” as used herein refers to a BACE binding site that is not theBACE active site. A BACE exosite is an important target site formodulating the processing of APP and the production of Aβ.

[0028] The present invention provides isolated peptides thatspecifically bind to BACE at an exosite and modulate BACE activity.Peptides that specifically bind to BACE at an exosite are also referredto herein as “exosite binding peptides” (EBPs). The terms “specificbinding” or “specifically bind” refer to the interaction between aprotein and a binding molecule, such as a compound. The interaction isdependent upon the presence of a particular structure (i.e., an enzymebinding site, an antigenic determinant or epitope) of the protein thatis recognized by the binding molecule. For example, if a compound isspecific for enzyme binding site “A”, the presence of the compound in areaction containing a protein including enzyme binding site A, and alabeled peptide that specifically binds to enzyme binding site A willreduce the amount of labeled peptide bound to the protein. In contrast,nonspecific binding of a compound to the protein does not result in aconcentration-dependent displacement of the labeled peptide from theprotein.

[0029] As used herein the term “active site” means the site on theenzyme molecule at which the chemical transformations of the substratemolecule take place. The term “exosite” as used herein means any site onthe enzyme molecule other than the active site.

[0030] The present invention provides a method for identifying peptidesthat specifically bind to a BACE exosite comprising:

[0031] (a) contacting BACE with at least one peptide; and

[0032] (b) determining whether the peptide specifically binds to BACE ata site other than the active site of BACE.

[0033] In one embodiment, the EBPs of the present invention can be usedto treat disorders such as neurodegenerative disorders. In thisembodiment, the EBP is administered to a patient in a therapeuticcomposition with a pharmaceutically acceptable carrier. Moreover, acombination of EBPs may be administered to a patient to treat aneurodegenerative disorder, such as Alzheimer's disease.

[0034] Peptides that bind to BACE exosites can be identified byscreening peptide libraries. Preferably, phage display random librariesand phage ELISA assays are used to identify the EBPs. Preparation ofphage display libraries and phage ELISA assays are known to thoseskilled in the art, see, for example, B. K. Kay, J. Winter, J.McCafferty (eds.), Phage Display of Peptides and Proteins. A LaboratoryManual, Academic Press, (1996), chapters 5, 7, 13 and 16. In a preferredembodiment, the peptides of the peptide libraries are 5 mer to 30 merpeptides. The phage display library can be screened by isolating viralparticles that bind to targets. The isolates can be grown up, and thedisplayed peptide sequence responsible for binding can be deduced by DNAsequencing.

[0035] In a preferred embodiment, the present invention provides EBPscomprising an amino acid sequence having a Tyr-Pro-Tyr-Phe (alsoreferred to herein as “YPYF”) (SEQ ID NO:1) motif wherein the EBPs arecapable of specifically binding to a BACE exosite and inhibiting BACEactivity.

[0036] Other preferred EBPs of the present invention include a BACEexosite binding motif comprising amino acid residues Tyr-Pro-Tyr-Phe-Ile(also referred to herein as “YPYFI”) (SEQ ID NO:2). Preferred EBPs ofthe present invention comprise at least one of the following amino acidsequences: Xaa-Tyr-Pro-Tyr-Phe (SEQ ID NO:3), Xaa-Tyr-Pro-Tyr-Phe-Xaa(SEQ ID NO:4), Xaa-Tyr-Pro-Tyr-Phe-Xaa-Xaa (SEQ ID NO:5),Tyr-Pro-Tyr-Phe-Xaa (SEQ ID NO:6) Tyr-Pro-Tyr-Phe-Xaa-Xaa (SEQ ID NO:7),His-Tyr-Pro-Tyr-Phe (SEQ ID NO:8), Tyr-Pro-Tyr-Phe-Ile (SEQ ID NO:2),Tyr-Pro-Tyr-Phe-Ile-Pro (SEQ ID NO:9), Tyr-Pro-Tyr-Phe-Ile-Pro-Leu (SEQID NO:10), Tyr-Pro-Tyr-Phe-Leu-Pro-Ile (SEQ ID NO:11),Tyr-Pro-Tyr-Phe-Xaa-Pro-Ile (SEQ ID NO:12), Tyr-Pro-Tyr-Phe-Xaa-Pro-Xaa(SEQ ID NO:13), His-Tyr-Pro-Tyr-Phe-Ile-Pro (SEQ ID NO:14)Tyr-Pro-Tyr-Phe-Leu (SEQ ID NO: 15), Tyr-Pro-Tyr-Phe-Leu-Pro (SEQ IDNO:16), His-Tyr-Pro-Tyr-Phe-Leu-Pro (SEQ ID NO:17), andHis-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu (SEQ ID NO:18). As used herein the term“Xaa” means any amino acid, i.e., either naturally or non-naturallyoccurring amino acid.

[0037] The most preferred EBPs of the present invention areAsn-Leu-Thr-Thr-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Pro (SEQ ID NO:19) alsoreferred to herein as “NLTTYPYFIPLP” and “BMS-561871”;Ala-Leu-Tyr-Pro-Tyr-Phe-Leu-Pro-Ile-Ser-Ala-Lys (SEQ ID NO:20) alsoreferred to herein as “ALYPYFLPISAK” and “BMS-561877”; andTyr-Pro-Tyr-Phe-Ile-Pro-Leu (SEQ ID NO:10) also referred to herein as“YPYFIPL” and “BMS-593925.”

[0038] Other preferred EBPs of the present invention comprise amino acidsequences having a WPXFI (SEQ ID NO:21) motif. Preferred EBPs having theWPXFI motif areGlu-Thr-Trp-Pro-Arg-Phe-Ile-Pro-Tyr-His-Ala-Leu-Thr-Gln-Gln-Thr-Leu-Lys-His-Gln-Gln-His-Thr(SEQ ID NO:22),Thr-Ala-Glu-Tyr-Glu-Ser-Arg-Thr-Ala-Arg-Thr-Ala-Pro-Pro-Ala-Pro-Thr-Gln-His-Trp-Pro-Phe-Phe-Ile-Arg-Ser-Thr(SEQ ID NO:23) and His-Trp-Pro-Phe-Phe-Ile-Arg-Ser (SEQ ID NO:57).

[0039] In the most preferred embodiment, the EBPs of the presentinvention contain from about 5 to about 30 amino acid residues.

[0040] The amino acid sequence of the subject EBPs can be modified forsuch purposes as enhancing therapeutic or prophylactic efficacy, orstability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Such modified peptide can be produced, forinstance, by amino acid substitution, deletion, or addition differentcodon usage. Likewise, different codons may be selected to increase therate at which expression of the peptide/polypeptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilized by the host.

[0041] Variant EBPs resulting from amino acid substitutions, deletions,or additions of the EBP sequences described herein are within the scopeof the present invention. Examples of such variant EBPs are EBPs whereina leucine is replaced with an isoleucine or valine, an aspartic acidwith a glutamic acid, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(i.e., conservative mutations). Conservative replacements are those thattake place within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into thefollowing families: (1) acidic: aspartatic acid, glutamatic acid; (2)basic: lysine, arginine, histidine; (3) nonpolar: alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4)uncharged polar: glycine, asparagine, glutamine, cysteine, serine,threonine, and tyrosine; (5) aromatic: phenylalanine, tryptophan, andtyrosine; (6) aliphatic: glycine, alanine, valine, leucine, isoleucine,serine, threonine, with serine and threonine optionally being groupedseparately as aliphatic-hydroxyl; and (7) amide: asparagine, glutamine;and (8) sulfur-containing: cysteine and methionine (see, for example,Stryer (ed.), Biochemistry, (2^(nd) ed.), W H Freeman and Co. (1981)).

[0042] The EBPs of the present invention can also be peptide mimicswherein one or more of the amino acid residues is replaced with anonnaturally occurring amino acid residue. For example, one or moreamino acid residues may be tagged with a photoaffinity label such as,for example, benzophenone.

[0043] Those skilled in the art of peptide chemistry are aware thatamino acid residues occur as both D and L isomers, and that the instantinvention contemplates the use of either D or L isomers or a mixture ofisomers of amino acid residues incorporated in the synthesis of thepeptides described herein.

[0044] The EBPs of the present invention can be produced by conventionalmethods known to those skilled in the art. In one embodiment, thepeptide may be produced by expression from a transformed host. Forexample, a host cell transfected with a nucleic acid vector directingexpression of a nucleotide sequence encoding the EBP can be culturedunder appropriate conditions to allow expression of the peptide tooccur. The peptide may be secreted and isolated from a mixture of cellsand medium containing the recombinant EBP. Alternatively, the peptidemay be retained cytoplasmically and the cells harvested, lysed and thepeptide isolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The recombinant EBP can be isolated from cell culture medium, hostcells, or both using techniques known in the art for purifying peptidesincluding ion-exchange chromatography, reverse phase chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for such peptides.In one embodiment of the invention, the recombinant EBP is a fusionprotein containing a domain that facilitates its purification, such asEBP-GST fusion protein.

[0045] In addition, cell-free translation systems (see Sambrook et al.,Molecular Cloning: A Laboratory Manual, (2^(nd) ed.) Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1989)) can be used to producerecombinant EBPs. Suitable cell-free expression systems for use inaccordance with the present invention include rabbit reticulocytelysate, wheat germ extract, canine pancreatic microsomal membranes, E.coli S30 extract, and coupled transcription/translation systems (PromegaCorp., Madison, Wis.). These systems allow expression of recombinantpolypeptides or peptides upon the addition of cloning vectors, DNAfragments, or RNA sequences containing coding regions and appropriatepromoter elements.

[0046] In another embodiment, nucleic acid sequences encoding the EBPsof the present invention may be synthesized, in whole or in part, usingchemical methods well known in the art (see, e.g., Caruthers, M. H. etal., (1980) Nucl. Acids Res. Symp. Ser. 215-223; Horn, T. et al., (1980)Nucl. Acids Res. Symp. Ser. 225-232). Such nucleic acid sequences can beexpressed by conventional methods known to those skilled in the art. Thepresent invention provides isolated codon-usage variants that do notalter the polypeptide sequence or biological activity of the EBPsdisclosed herein. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms may occur due to degeneracy in the genetic code. Examplesinclude nucleotide codons CGT, CGG, CGC, and CGA encoding the aminoacid, arginine (R); or codons GAT, and GAC encoding the amino acid,aspartic acid (D). Thus, a protein or peptide can be encoded by one ormore nucleic acid molecules that differ in their specific nucleotidesequence, but still encode peptide or protein molecules having identicalsequences. The amino acid coding sequence is as follows: Three One AminoLetter Letter Acid Symbol Symbol Codons Alanine Ala A GCU, GCC, GCA, GCGCysteine Cys C UGU, UGC Aspartic Asp D GAU, GAC Acid Glutamic Glu E GAA,GAG Acid Phenylal- Phe F UUU, UUC anine Glycine Gly G GGU, GGC, GGA, GGGHistidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Lysine Lys KAAA, AAG Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG Methionine Met M AUGAsparagine Asn N AAU, AAC Proline Pro P CCU, CCC, CCA, CCG Glutamine GlnQ CAA, CAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU,UCC, UCA, UCG, AGU, AGC Threonine Thr T ACU, ACC, ACA, ACG Valine Val VGUU, GUC, GUA, GUG Tryptophan Trp W UGG Tyrosine Tyr Y UAU, UAC

[0047] The codon-usage variants may be generated by recombinant DNAtechnology. Codons may be selected to optimize the level of productionof the EBP in a particular prokaryotic or eukaryotic expression host, inaccordance with the frequency of codon utilized by the host cell.Alternative reasons for altering the nucleotide sequence encoding an EBPinclude the production of RNA transcripts having more desirableproperties, such as an extended half-life or increased stability. Amultitude of variant nucleotide sequences that encode the respectiveEBPs may be isolated, as a result of the degeneracy of the genetic code.Accordingly, the present invention provides selecting every possibletriplet codon to generate every possible combination of nucleotidesequences that encode the disclosed EBPs.

[0048] Alternatively, the peptide or protein itself may be producedusing chemical methods to synthesize the amino acid sequence of the EBP,or a portion thereof. For example, peptide synthesis can be performedusing various solid-phase techniques (see, e.g., Roberge, J. Y. et al.,(1995) Science 269:202-204), cleavage from a naturally-derived,synthetic or semi-synthetic polypeptide, automated synthesis using apeptide synthesizer, or a combination of these techniques.

[0049] Solid-phase techniques that can be used to synthesize the EBPs ofthe present invention are described in G. Barany and R. B. Merrifield,The Peptides: Analysis, Synthesis, Biology; Volume 2—“Special Methods inPeptide Synthesis, Part A”, pp. 3-284, (E. Gross and J. Meienhofer,eds.), Academic Press, New York, 1980; and in J. M. Stewart and J. D.Young, Solid-Phase Peptide Synthesis, 2^(nd) Ed., Pierce Chemical Co.,Rockford, Ill., (1984), for example. The preferred strategy for use inthis invention is based on the Fmoc (9-Fluorenylmethylmethyloxycarbonyl)group for temporary protection of the α-amino group, in combination withthe tert-butyl group for temporary protection of the amino acid sidechains (see for example E. Atherton and R. C. Sheppard, “TheFluorenylmethoxycarbonyl Amino Protecting Group”, in The Peptides:Analysis, Synthesis, Biology; Volume 9—“Special Methods in PeptideSynthesis, Part C”, pp. 1-38, (S. Undenfriend and J. Meienhofer, eds.),Academic Press, San Diego, (1987)).

[0050] The peptides are synthesized in a stepwise manner on an insolublepolymer support (also referred to as “resin”) starting from theC-terminus of the peptide. A synthesis is begun by appending theC-terminal amino acid of the peptide to the resin through formation ofan amide linkage. This allows the eventual release of the resultingpeptide as a C-terminal amide. The C-terminal amino acid and all otheramino acids used in the synthesis are required to have their α-aminogroups and side chain functionalities (if present) differentiallyprotected such that the α-amino protecting group may be selectivelyremoved during the synthesis. The coupling of an amino acid is performedby activation of its carboxyl group as an active ester and reactionthereof with the unblocked α-amino group of the N-terminal amino acidappended to the resin. The sequence of α-amino group deprotection andcoupling is repeated until the entire peptide sequence is assembled. Thepeptide is then released from the resin with concomitant deprotection ofthe side chain functionalities, usually in the presence of scavengers tolimit side reactions. The resulting peptide is finally purified byreverse phase HPLC.

[0051] The synthesis of the peptidyl-resins required as precursors tothe final EBP peptides utilize commercially available cross-linkedpolystyrene polymer resins. Preferred for use in this invention is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methylbenzhydrylamine resin (Rink amide MBHA resin), Novabiochem, San Diego,Calif. Coupling of amino acids can be accomplished using HOBT or HOATactive esters produced from HBTU/HOBT in the presence of a tertiaryamine such as DIEA, or from DIC/HOAT, respectively.

[0052] Preferred Fmoc amino acids for use in synthesizing the EBPs ofthe present invention are the derivatives shown below.

[0053] Orthogonally Protected Amino Acids Used in Solid Phase Synthesis

[0054] The peptidyl-resin precursors for their respective peptides maybe cleaved and deprotected using any of the standard proceduresdescribed in the literature (see, for example, King et al., (1990) Int.J. Peptide Protein Res. 36:255-266). A preferred method for use in thisinvention is the use of TFA in the presence of water and TIS asscavengers. Typically, the peptidyl-resin is stirred in TFA/water/TIS(94:3:3, v:v:v; 1 mL/100 mg of peptidyl resin) for 1.5-2 hrs at roomtemperature. The spent resin is then filtered off and TFA solution isconcentrated or dried under reduced pressure. The resulting crudepeptide is either washed with Et₂O or redissolved directly into DMSO or50% aqueous acetic acid for purification by preparative HPLC.

[0055] Peptides with the desired purity can be obtained by purificationusing preparative HPLC on, for example, either a Waters Model 4000 or aShimadzu Model LC-8A liquid chromatograph. The solution of crude peptideis injected into a YMC S5 ODS (20×100 mm) column and eluted with alinear gradient of MeCN in water, both buffered with 0.1% TFA, using aflow rate of 14-20 mL/min with effluent monitoring by UV absorbance at220 nm. The structures of the purified peptides are typically confirmedby electro-spray MS analysis.

[0056] Attachment of a fluorescent label to the EBP peptides describedherein may be accomplished by reacting either the α-amino group of theN-terminal amino acid residue of the EBP peptide or the ω-amino group ofthe side chain of a α,ω-diamino acid appended to the C-terminus of a EBPpeptide with the N-hydroxysuccinimidyl ester derivatives of the desiredfluorophore. Preferred for use in this invention is the Alexa Fluor 488fluorophore (“Alexa488”) (Molecular Probes, Eugene, Oreg.).

[0057] The following abbreviations are employed in the Examples andelsewhere herein:

[0058] TMS=trimethylsilyl; FMOC=fluorenylmethoxycarbonyl; Boc orBOC=tert-butoxycarbonyl; Bpa=p-benzoyl phenylalanine; HOAc orAcOH=acetic acid; MeCN=acetonitrile; DMF=N,N-dimethylformamide;TFA=trifluoroacetic acid; TIS=Triisopropylsilane; Et₂O=diethyl ether;NMP=N-methylpyrrolidone; DCM=dichloromethane;HOBT=1-hydroxybenzotriazole; HOAT=1-hydroxy-7-azabenzotriazole;HBTU=2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; DIC=N,N′-diisopropylcarbodiimide;DIEA=N,N-diisopropylethylamine; min=minute(s); h or hr=hour(s); L=liter;mL=milliliter; μL=microliter; g=gram(s); mg=milligram(s); mol=mole(s);mmol=millimole(s); meq=milliequivalent; rt=room temperature; sat orsat'd=saturated; aq.=aqueous; HPLC=high performance liquidchromatography; LC/MS=high performance liquid chromatography/massspectrometry; MS or Mass Spec=mass spectrometry.

[0059] In accordance with the present invention, isolated and/orsynthetic EBPs can also be used to identify BACE exosites, and are auseful tool for characterizing the structure of BACE exosites. Forexample, a BACE exosite may be characterized by crosslinking an EBPtagged with a photoaffinity group or photoaffinity label to the BACEexosite. The terms “photoaffinity group” and “photoaffinity label” referto a substituent on the inhibitor which can be activated by photolysisat an appropriate wavelength to undergo a crosslinking photochemicalreaction with BACE. An example of a “photoaffinity group” is abenzophenone substituent.

[0060] In another embodiment of the present invention, the EBPs can beused as a BACE probe.

[0061] The following definitions apply to the terms used throughout thisspecification, unless otherwise defined in specific instances:

[0062] The term “BACE” as used herein refers to all forms of BACE,including BACE variants and proteins including the catalytic domain ofBACE, or a fragment of BACE containing a BACE exosite. A representative,but non-limiting, example of BACE is a protein encoded by all or afragment of the nucleic acid of GenBank Accession No. NM012104.

[0063] “Modulator of BACE” or “BACE modulator” as used herein refers toa compound that alters the activity of BACE, such as, for example,agonists that increase the activity of BACE or antagonists that inhibitthe activity of BACE.

[0064] The term “compound” as used herein includes but is not limited tosmall molecules, peptides, nucleic acid molecules and antibodies.

[0065] As used herein, “candidate modulator of BACE” is intended to meanany compound that can be screened for activity to inhibit BACE using theassay of the invention described herein. It is understood that a“candidate modulator of BACE”, which is active in the assay of theinvention for inhibiting BACE activity, can subsequently be used as a“BACE modulator” or “BACE inhibitor”. It is also understood that a“candidate modulator of BACE”, which is active in the assay of theinvention for inhibiting BACE activity, can subsequently be used inpharmaceutical compositions for the treatment of degenerativeneurological disorders involving beta-amyloid production, preferably forthe treatment of Alzheimer's disease.

[0066] As used herein, “candidate inhibitor of beta-amyloid production”is intended to mean any compound that can be screened for activity toinhibit the production of beta-amyloid peptide, or the proteolyticactivity leading to the production of beta-amyloid peptide, using theassay of the invention described herein. It is understood that a“candidate inhibitor of beta-amyloid production”, which is active in theassay of the invention for inhibiting the production of beta-amyloidpeptide, can subsequently be used as a “beta-amyloid peptide inhibitor.”It is also understood that a “candidate inhibitor of beta-amyloidproduction”, which is active in the assay of the invention forinhibiting the production of beta-amyloid peptide, can subsequently beused in pharmaceutical compositions for the treatment of degenerativeneurological disorders involving beta-amyloid production, preferably forthe treatment of Alzheimer's disease.

[0067] The “inhibitory concentration” of a BACE modulator or inhibitoris intended to mean the concentration at which a compound screened in anassay of the invention inhibits a measurable percentage of BACEactivity. Examples of “inhibitory concentration” values range from IC₅₀to IC₉₀, and are preferably, IC₅₀, IC₆₀, IC₇₀, IC₈₀, or IC₉₀, whichrepresent 50%, 60%, 70%, 80% and 90% reduction in BACE activity,respectively. More preferably, the “inhibitory concentration” ismeasured as the IC₅₀ value. It is understood that another designationfor IC₅₀ is the half-maximal inhibitory concentration.

[0068] Likewise, as used herein, “inhibitory concentration” of abeta-amyloid production inhibitor is intended to mean the concentrationat which a compound screened in an assay of the invention inhibits ameasurable percentage of beta-amyloid peptide production. Examples of“inhibitory concentration” values range from IC₅₀ to IC₉₀, and arepreferably, IC₅₀, IC₆₀, IC₇₀, IC₈₀, or IC₉₀, which represent 50%, 60%,70%, 80% and 90% reduction in beta-amyloid peptide production,respectively. More preferably, the “inhibitory concentration” ismeasured as the IC₅₀ value. It is understood that another designationfor IC₅₀ is the half-maximal inhibitory concentration.

[0069] The EBPs of the present invention are particularly useful foridentifying inhibitors of Aβ production. The EBPs can be used incompetitive binding assays to identify inhibitors of proteolyticactivity leading to Aβ production for the treatment of neurologicaldisorders, such as Alzheimer's disease, Down's syndrome and otherdisorders involving Aβ, APP, and/or Aβ/APP associated macromolecules.Such competitive binding assays can identify compounds that interferewith the binding of EBPs to isolated BACE, complexes of BACE and othermacromolecules, relevant tissues, cell lines, and membranes derived fromrelevant tissues and cell lines.

[0070] In one embodiment, the present invention provides a method foridentifying modulators of BACE comprising the steps of:

[0071] (a) contacting a candidate modulator of BACE and an exositebinding peptide (EBP) in the presence of a BACE including at least oneBACE exosite; and

[0072] (b) determining whether there is a decrease in binding of theexosite binding peptide to BACE in the presence of the candidate BACEmodulator compared to binding of the exosite binding peptide to BACE inthe absence of the candidate modulator.

[0073] The binding to and displacement from BACE of exosite bindingpeptides (EBPs) can be determined by methods well known to those skilledin the art. The form of BACE used for such experiments can berecombinant or natural full length BACE within the environment of acellular membrane, or solubilized from a membrane by appropriatetreatment with a detergent. Alternatively, the purified, recombinantcatalytic domain of BACE can be used in the binding measurements. BACEmolecules such as for example, allelic variants, fragments, or fusionproteins including at least one BACE exosite of interest are within thescope of the invention for use in the screening assays herein. In apreferred embodiment of the present invention BACE is recombinant humanBACE catalytic domain as described in Mallender et al., (2001) Mol.Pharmacol. 59:619-626, and as described herein in Example 3.

[0074] Binding of the EBPs to BACE can be measured, for example, bymethods such as isothermal titration calorimetry, nuclear magneticresonance spectroscopy, BIAcore technology and the like. In a preferredembodiment, the EBPs can be modified by the incorporation of achromophoric, fluorophoric or radioactive species to provide aconvenient label with which to follow the interactions of the peptideswith the macromolecular enzyme. As an example, a fluorescent moleculecan be covalently attached to the amino terminus, to the carboxylterminus, or to specific amino acid side chains (e.g., lysines andcysteines) of the peptide by application of standard peptide chemistrythat is well known to those skilled in the art. For example, the EBP canbe labeled with Alexa488 (Molecular Probes, Eugene, Oreg.). Once labeledand purified, the now fluorescent EBP can be conveniently used tomeasure formation of a binary complex with the BACE molecule.

[0075] In one aspect of the present invention, the fluorescent EBP canbe mixed with BACE under conditions that optimally promote binding, fora sufficient time to establish an equilibrium between the bound and freeforms of the enzyme and peptide. The free peptide can then be rapidlyseparated from the enzyme-bound population by any of several methodsthat effect separation of molecules based on molecular mass, such as gelfiltration chromatography, dialysis and membrane filtration. The amountof fluorescent EBP associated with the enzyme can then be quantified byfluorescence spectroscopy. By measuring the concentration of EBP boundto the enzyme as a function of enzyme and EBP concentration, theequilibrium dissociation constant, K_(d), for the enzyme-EBP binarycomplex can be determined by standard methods well known to thosetrained in the art (see, for example, Copeland, R. A., Enzymes: APractical Introduction to Structure, Mechanism and Data Analysis,(2^(nd) ed.), Wiley-VCH, New York, N.Y. (2000)). Having determined theK_(d), one can mix a specific concentration of BACE and EBP to establisha particular level of EBP occupancy on BACE. Addition of compounds thatcompete with EBPs for binding to BACE would cause a shift in thefractional occupancy of the fluorescent EBP on BACE. By measuring theshift in fractional occupancy as a function of the concentration ofcompeting compound, one can define the K_(d) of the competing compoundby methods well known to those skilled in the art (see, for example,Copeland, R. A., Enzymes: A Practical Introduction to Structure,Mechanism and Data Analysis, (2^(nd) ed.), Wiley-VCH, New York, N.Y.(2000)).

[0076] Often, the fluorescence properties of a molecule will change uponcomplex formation with a protein. Hence, changes in a fluorescenceemission wavelength maximum or fluorescence intensity may accompanybinding of the labeled EBP to BACE. In such cases, the change influorescence property can be used as a direct measure of binding,without the need to physically separate the bound and free populationsof labeled EBP.

[0077] In a preferred embodiment, the polarization (or anisotropy) offluorescence is measured with a suitable instrument. The degree offluorescence polarization depends on the rotational freedom of thefluorescent molecule. When free in solution the fluorescencepolarization of the labeled EBP would have a characteristic low value.Upon complexation with BACE, the rotational freedom is diminished andthe degree of fluorescence polarization increases markedly. Thesechanges in characteristic fluorescence polarization can therefore beused to measure the fractional occupancy of EBPs on BACE and, asdescribed above, can also be used to measure the binding affinity ofcompeting molecules. In a manner similar to that described above, afixed concentration mixture of BACE and labeled EBP is mixed withvarying concentrations of a competing compound. Displacement of the EBPcaused by competition with the compound for the binding site on BACE isquantified by the changes in fluorescence polarization value.

[0078] Alternatively, a fluorescent or chromophoric molecule can becovalently associated with the BACE enzyme through standard proteinchemistry methods that are well known to those skilled in the art. Thespectroscopic features of the molecule are chosen to overlap those of afluorescent group attached to the EBP as described above, such that theabsorbance maximum of the species attached to the enzyme overlaps thefluorescence maximum of the species attached to the EBP. When the enzymeand EBP are separate, the maximal fluoresence of the species attached tothe EBP is realized. However, when the binding of the EBP to BACE bringsthe spectroscopic species attached to BACE and the EBP into proximity,the overlap of spectral properties will cause a diminution offluorescence intensity for the group attached to the EBP in what iscommonly referred to as Fluorescence Resonance Energy Transfer (FRET).The diminution of fluorescence intensity that accompanies bindingbetween BACE and the EBP can be directly quantified as a measure ofbinding interactions. The addition of a competing molecule to a mixtureof the BACE/EBP FRET pair would cause a relief of the fluorescenceintensity quenching which could thus be used to measure competitivebinding of compounds to the EBP binding site on BACE.

[0079] In yet another embodiment, the EBP is labeled by incorporation ofa radioactive species, such as ³H, ¹⁴C, ³⁵S, ³³P, ¹²⁵I, etc., bystandard methods of peptide chemistry. In a manner similar to thatdescribed above, the binding of the radiolabeled EBP to BACE can befollowed by mixing the peptide and protein together under optimalconditions and then rapidly separating the free peptide population fromthe enzyme-bound population.

[0080] In a further embodiment of the present invention an affinitysequence can be appended to the amino acid sequence of the BACE enzymeusing standard methods of recombinant DNA technology. Examples of suchaffinity sequences include, but are not limited to multiple histidineresidues for complexation with transition metals, epitopic sequencesthat are recognized by specific antibodies, and biotin which isrecognized by the protein streptavidin. Technology well known to thoseskilled in the art commonly referred to as a Scintillation ProximityAssay (SPA) can be used to measure binding of the labeled EBP to BACEand the displacement of this binding by competing molecules.

[0081] Polymeric beads that are saturated with scintillation fluid andare chemically attached to the recognition partner of the affinitysequence, i.e., chemically attached to a transition metal, a specificantibody, or to streptavidin or other recognition partners, can be mixedwith the BACE protein containing the affinity sequence to form a stablecomplex between the BACE protein and the polymeric bead. Whenradiolabeled EBP is added to this mixture, the binding of the EBP toBACE brings the radiolabel on the peptide into close proximity with thescintillation fluid incorporated into the polymeric bead. The resultinglight emission from the scintillation fluid can then be quantified as ameasure of binding interaction between BACE and the peptide. In a mannersimilar to that outlined above, the signal measured in this way can beused to quantify binding of the labeled EBP to BACE and the displacementof this binding by competing molecules.

[0082] Any of the above methods can be adapted for use in highthroughput screening of compound libraries to discover molecules thatcompete with the EBP for binding to the exosite on BACE. Standardmethods can be used to adapt the methods described above formeasurements in micro-well plates of varying formats including, but notlimited to, 96, 384 and 1536 wells per plate. In a common highthroughput screening application, the BACE enzyme and EBP are mixed atfixed concentrations in each well of the micro-well plate. To individualwells of each plate is added one compound of a compound library at afixed concentration. After mixing the signal associated with BACE/EBPcomplex formation is measured by use of an appropriate microplatereading instrument. Library compounds that alter the signal associatedwith BACE/EBP complex formation can thus be identified as potentialcompetitors for the EBP binding site on BACE. These library compoundscan then be characterized further to determine their individual bindingaffinity for BACE by the more complete methods described above.

[0083] The present invention provides a method for identifyinginhibitors as therapeutics for disorders involved in APP processing andbeta-amyloid production comprising:

[0084] (a) contacting BACE with a candidate BACE exosite bindingcompound; and

[0085] (b) determining the amount of inhibition of APP processing andbeta-amyloid production.

[0086] The present invention provides a cell based assay for identifyingBACE exosite binding compounds that inhibit beta-amyloid productioncomprising:

[0087] (a) contacting a candidate BACE exosite binding compound with acell that expresses a beta amyloid precursor protein and BACE whereinthe cell is capable of secreting beta-amyloid protein in the absence ofthe candidate exosite binding compound; and

[0088] (b) determining whether the candidate exosite binding compoundreduces the amount of beta amyloid protein secreted by the cell.

[0089] In another embodiment of the invention, the method foridentifying BACE exosite binding compounds that inhibit beta-amyloidproduction is performed using cell membranes or in a cell-free settingusing cell-free enzyme and cell-free substrate according to methodsknown to those skilled in the art.

[0090] The present invention provides EBPs that bind to a BACE exositeand inhibit BACE activity. Inhibition of BACE activity by the EBPs ofthe present invention can be demonstrated using beta-amyloid precursorprotein (also referred to herein as “β-APP” or “APP”), the precursor forAβ, which through the activity of secretase enzymes is processed intoAβ. Secretase enyzmes known in the art have been designated β secretase,which generates the N-terminus of Aβ, α secretase cleaving around the16/17 peptide bond in Aβ, and γ secretase which generates C-terminal Aβfragments ending at position 38, 39, 40, 41, 42, and 43, or C-terminalextended precursors which are subsequently truncated to the abovepeptides.

[0091] In accordance with the present invention, full length human APP,known mutations thereof (e.g., the Swedish mutant), fragments of humanwild type or mutant APP, peptides derived from human wild type or mutantAPP as well as APP or APP fragments fusion proteins, such as, MBP-APP(which includes APP residues 547-595) can be used as a substrate toconfirm inhibition of BACE activity by an EBP. The peptide bondhydrolysis activity of BACE can be determined by contacting anappropriate substrate with the enzyme under optimized reactionconditions and then measuring the loss of substrate or production ofhydrolysis products as a function of reaction time by some suitableanalytical detection method. For example, the recombinant catalyticdomain of human BACE can be incubated with the peptideMCA-EVNLDAEFK(-dnp)-COOH (SEQ ID NO:107) in which MCA is a7-methoxycoumarin-4-acetyl group and dnp is a dinitrophenyl groupappended to the epsilon amino group of the lysine side chain. Thispeptide sequence reflects the amino acid sequence surrounding the betacleavage site of Swedish mutant APP. The MCA group is highly fluorescentbut its fluorescence is quenched by proximity to the dnp group. Thus,the peptide displays low fluorescence signal when intact, but thefluorescence signal is greatly augmented upon BACE-mediated hydrolysisof the peptide.

[0092] After a fixed time of incubation, the increase in fluorescencesignal can be used as a measure of BACE activity, as described morefully in Mallender et al., (2001) Mol. Pharmacol. 59:619-626 and inMarcinkeviciene et al., (2001) J. Biol. Chem. 276, 23790-23794. Theability of an EBP to inhibit the BACE-mediated hydrolysis of thissubstrate would be reflected in a diminished fluorescence signal aftersubstrate incubation with BACE in the presence of the EBP.

[0093] Such assays can be performed in a cell free setting, usingcell-free enzyme and cell-free substrate, or can be performed in acell-based assay, or using cell membranes according to methods known tothose skilled in the art.

[0094] The present invention further provides a method of treating aneurological disorder comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of a compound thatinhibits beta-amyloid production, or a pharmaceutically acceptable saltor prodrug form thereof, wherein the compound binds to a BACE exositeand effects a decrease in production of beta-amyloid.

[0095] The compounds determined from the present invention can beadministered orally using any pharmaceutically acceptable dosage formknown in the art for such administration. The active ingredient can besupplied in solid dosage forms such as dry powders, granules, tablets orcapsules, or in liquid dosage forms, such as syrups or aqueoussuspensions. The active ingredient can be administered alone, but isgenerally administered with a pharmaceutical carrier. A valuabletreatise with respect to pharmaceutical dosage forms is Remington'sPharmaceutical Sciences (17^(th) ed.), Mack Publishing Co., Easton, Pa.,(1985).

[0096] The compounds determined from the present invention can beadministered in such oral dosage forms as tablets, capsules (each ofwhich includes sustained release or timed release formulations), pills,powders, granules, elixirs, tinctures, suspensions, syrups, andemulsions. Likewise, they may also be administered in intravenous (bolusor infusion), intraperitoneal, subcutaneous, or intramuscular form, allusing dosage forms well known to those of ordinary skill in thepharmaceutical arts. An effective but non-toxic amount of the compounddesired can be employed to prevent or treat neurological disordersrelated to beta-amyloid production or accumulation, such as Alzheimer'sdisease and Down's Syndrome.

[0097] The compounds of this invention can be administered by any meansthat produces contact of the active agent with the agent's site ofaction in the body of a host, such as a human or a mammal. They can beadministered by any conventional means available for use in conjunctionwith pharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

[0098] The dosage regimen for the compounds determined from the presentinvention will, of course, vary depending upon known factors, such asthe pharmacodynamic characteristics of the particular agent and its modeand route of administration; the species, age, sex, health, medicalcondition, and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment; the frequency of treatment;the route of administration, the renal and hepatic function of thepatient, and the effect desired. An ordinarily skilled physician orveterinarian can readily determine and prescribe the effective amount ofthe drug required to prevent, counter, or arrest the progress of thecondition.

[0099] Advantageously, compounds determined from the present inventionmay be administered in a single daily dose, or the total daily dosagemay be administered in divided doses of two, three, or four times daily.

[0100] The compounds identified using the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

[0101] In the methods of the present invention, the compounds hereindescribed in detail can form the active ingredient, and are typicallyadministered in admixture with suitable pharmaceutical diluents,excipients, or carriers (collectively referred to herein as carriermaterials) suitably selected with respect to the intended form ofadministration, that is, oral tablets, capsules, elixirs, syrups and thelike, and consistent with conventional pharmaceutical practices.

[0102] For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as lactose,starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral drug components can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor β-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like.

[0103] The compounds determined from the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamallar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine, or phosphatidylcholines.

[0104] Compounds of the present invention may also be coupled withsoluble polymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues.

[0105] Furthermore, the compounds determined from the present inventionmay be coupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

[0106] Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivatives, magnesiumstearate, stearic acid, and the like.

[0107] Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, or entericcoated for selective disintegration in the gastrointestinal tract.Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. In general, water, a suitableoil, saline, aqueous dextrose (glucose), and related sugar solutions andglycols such as propylene glycol or polyethylene glycols are suitablecarriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences (1985).

EXAMPLES

[0108] The following examples as set forth herein are meant toillustrate and exemplify the various aspects of carrying out the presentinvention and are not intended to limit the invention in any way.

[0109] The synthetic peptides described herein were prepared asN-terminal acetyl derivatives and as C-terminal carboxy amides, with theexception of those peptides identified as SEQ ID NOs:19 and 48, whichwere prepared as N-terminal acetyl derivatives but did not contain aC-terminal carboxy amide group.

Example 1

[0110] BACE Exosite Binding Peptides from Solid Phase Panning at pH 7.0.

[0111] Two highly selected and homologous 12 mer phage peptides boundBACE specifically and reproducibly in phage-ELISA tests. Bristol-MyersSquibb fUSE5-based C4C, C6C, 5- and 15 mer libraries, and M13-based C7Clibraries, and 7- and 12 mer libraries obtained from New EnglandBiolabs, Beverly, Mass. were panned for three cycles against BACE(produced as described herein in Example 3). BACE was immobilized bycoating at 0.5 μg/well in 4 wells of Dynex Immulon 4HBX plates overnightat 4° C. in 0.1M NaHCO₃ buffer, pH 9.0. Panning was by standardprocedures at room temperature that involved blocking wells with 2% BSAin PBS and elution with 0.1M HCL, pH 2.2. The vector NTI alignment tooland visual inspection of sequences were employed to analyze the selectedpeptides.

[0112] After sequencing approximately 20-50 clones from each libraryafter three cycles of selection, we prepared essentially all possiblecandidate clones (39 clones in total) for phage-ELISA to obtain directevidence for affinity to BACE. Eleven clones gave binding signals andone of those clones, a 12 mer clone (NLTTYPYFIPLP (SEQ ID NO:19)), wasreproducibly shown to specifically bind to BACE. We therefore sequencedadditional 12 mer clones to try to find additional candidate clones.Eleven candidate clones were tested by phage ELISA and one clone(ALYPYFLPISAK (SEQ ID NO:20)) exhibited specific binding to BACE. ThisALYPYFLPISAK (SEQ ID NO:20) peptide is homologous to NLTTYPYFIPLP (SEQID NO:19) and consistent with the specific binding of both thosepeptides to BACE. The ALYPYFLPISAK (SEQ ID NO:20) peptide andNLTTYPYFIPLP (SEQ ID NO:19) peptides were the two most efficientlyrecovered clones with 13 and 9 copies, respectively.

Example 2

[0113] BACE Binding Peptides from Solid- and Solution Phase Panning atpH 5.0.

[0114] Solid phase panning experiments at pH 7.0 yielded exosite BACEbinding peptides BMS-561871 and BMS-561877 which share a conserved coreregion. Solution and solid phase panning at pH 5.0 yielded 21 peptideswith essentially the same conserved core region that is present inBMS-561871 and BMS-561877. Overall, solution phase panning appeared tofacilitate the isolation of these peptides. This is consistent with theidea that the peptide binding site on BACE may be less accessible whenBACE is immobilized, as in solid phase panning. The presence of theactive site inhibitor OM99-2 in solid phase panning did not noticeablyimprove the ability to recover these peptides. In the absence of OM99-2,any selection for peptides that occupy the active site of BACE maytherefore be less efficient or absent. The results are consistent withthe idea that the new set of peptides binds BACE outside the activesite.

[0115] Phage ELISA indicated that all 21 peptides from the solid phasepanning bind BACE specifically at pH 5.0 and pH 7.0. Binding specificityfor peptides from solution phase was only tested at pH 5.0 and wheresignals were obtained (subject to phage concentrations which were notstandardized), peptides bound specifically. Consensus peptides areprovided at the end of Table 1. The presence of a histidine residueimmediately flanking the YPYF (SEQ ID NO:1) motif, i.e. HYPYF (SEQ IDNO:8), appears to contribute to more efficient binding at pH 5.0.BMS-561871 and BMS-561877 were isolated at pH 7.0 and lack histidine atthis position.

Peptides with Other Sequence Motifs

[0116] Two tight-binding peptides contained a conserved region that issignificantly different from, but clearly related to the core region inBMS-561871 and BMS-561877. Solid- and solution phase panning yielded 6and 4 groups of peptides, respectively, that contained motifs other thanthe YPYF motif in the peptides listed in Table 1. Two peptides fromsolution panning, ETWPRFIPYHALTQQTLKHQQHT (SEQ ID NO:22) andTAEYESRTARTAPPAPTQHWPFFIRST (SEQ ID NO:23), exhibited strong andspecific binding that was similar to phage carrying BMS-561871. TheETWPRFIPYHALTQQTLKHQQHT (SEQ ID NO:22) and TAEYESRTARTAPPAPTQHWPFFIRST(SEQ ID NO:23) BMS-561871 peptides include a WPXFI (SEQ ID NO:21) motif.The result is consistent with the fact that the two peptidesETWPRFIPYHALTQQTLKHQQHT (SEQ ID NO:22) and TAEYESRTARTAPPAPTQHWPFFIRST(SEQ ID NO:23) from solution panning share a region with homology thatis different from, but clearly similar to, the core region of thepeptides ALYPYFLPISAK (SEQ ID NO:20) and NLTTYPYFIPLP (SEQ ID NO:19).Thus, peptides containing the YPYF (SEQ ID NO:1) motif or closelyrelated sequences are able to efficiently bind BACE at the BACE exosite.

[0117] Panning

[0118] a.) Solid Phase:

[0119] The M13-based C7C-, 12-, and 15 mer libraries were panned in thepresence or absence of 1 μM OM99-2 against BACE produced as BACE-IgG₁from CHO cells and treated to remove the Ig-domain, as described inExample 3. Three panning cycles were carried out: BACE was immobilizedat 0.5 μg/well in 4 wells of Dynex Immulon plates overnight in 0.1MNaHCO₃ buffer, pH 9.0. This was followed by blocking the wells withPBS+2% BSA for 1 hour. For panning at pH 5.2, blocking buffer wasdiscarded and library phage was then added for two hours in 50 mM NaOAc,pH 5.2+2% BSA, followed by washes with 50 mM NaOAc, pH 5.2, +0.2% Tween20 and subsequent elution with 0.1M HCl, pH 2.2 for amplification or DNAsequencing after round three. For panning at pH 7.0, all buffers werebased on PBS instead of NaOAc.

[0120] b.) Solution Phase:

[0121] The following mixtures of our M13-based libraries were pannedagainst BACE-prepared from CHO cells (vide supra): a.) C7C+C8Clibraries, b.) 12-+15 mer libraries, and c.) 23-+27-+33 mer libraries.Library mixtures and BACE were pre-blocked in 50 mM NaOAc, pH 5.2, +2%BSA and then mixed for two hours. The mixtures were then added toPansorbin Protein A cells (Calbiochem) in 50 mM NaOAc, pH 5.2, afterblocking the cells in NaOAc, pH 5.2, plus 2% BSA and 1% milk. This stepwas followed by washing the Pansorbin cell-phage complexes several timeswith 50 mM NaOAc, pH 5.2, plus 0.2% Tween 20. Phage were eluted with 6Murea, pH 3.0, and used for amplification and further panning cycles orDNA sequencing after round three.

[0122] Phage ELISA

[0123] Standard procedures were used: BACE (without IgG, domain) wascoated in 0.1M NaHCO₃, pH 9.0 overnight at 4° C. From this pointonwards, all incubation and wash buffers were based on 50 mM NaOAc, pH5.2, for determining binding at pH 5.2. To determine binding at pH 7.0,NaOAc was replaced by PBS. TABLE 1 Peptides Having a YPYF MotifSpecifically Bind to a BACE Exosite SOLID PHASE PANNING AT pH 7.0:              NLTTYPYFIPLP (BMS-561871) (SEQ ID NO:19)                ALYPYFLPISAK (BMS-561877) (SEQ ID NO:20) SOLID PHASEPANNING AT pH 5.0:                QNHYPYFIAVPI (SEQ ID NO:24)             EGNKHYPYFIKV (SEQ ID NO:25)               THSHYPYFIELE (SEQID NO:26)                 QQYPYFIPVIRP (SEQ ID NO:27) SOLUTION PANNINGAT pH 5.0:                  HYPYFLPLHTPK (SEQ ID NO:28)    AMLDGAPTNRNSQHYPYFLPIATV (SEQ ID NO:29)       LPVYDTTAPTHYPYFLPLPRISP (SEQ ID NO:30)              EGNKHYPYFIKV(SEQ ID NO:25)              SQLQHYPYFRPL (SEQ ID NO:31)              YIPHYPYFIRLN (SEQ ID NO:32)      KMHSMINQLGTRHYPYFREINDY(SEQ ID NO:33)              GSTKSYPYFIHT (SEQ ID NO:34)     DIWNGAKAPKNSMYPYFIPSSLK (SEQ ID NO:35) ISVINQPAQNMHPRQMTAYPYFRPISR(SEQ ID NO:36)                 DVYPYFVSSNEGHSIRHKGNNSL (SEQ ID NO:37)                  YPYFIDSHPPKELMPHSWVQSKYPASPQTHTTY (SEQ ID NO:38)                 GYPYFLNLKNSH (SEQ ID NO:39)                NSYPYFIHLSNP (SEQ ID NO:40)                 HDYPYFMMLTGH(SEQ ID NO:41)               QIETYPYFLPIL (SEQ ID NO:42)                 YYPYFISTAREV (SEQ ID NO:43) Consensus:                 HYPYFIPL (SEQ ID NO:18)                  Y    L I                 T    V V                  S    M

[0124] TABLE 2 Peptides with Other Sequence Motifs SOLUTION PHASEPANNING AT pH 5.0:                  ETWPRFIPYHALTQQTLKH (SEQ ID NO:22)TAEYESRTARTAPPAPTQHWPFFIRST (SEQ ID NO:23)

Example 3

[0125] Isothermal Titration Calorimetry (ITC) of BACE and ExositePeptides

[0126] Isothermal titration calorimetry was performed to determinequantitatively the binding affinities of BMS-561877 and BMS-561871 forβ-secretase. Recombinant human BACE was expressed as a fusion proteinwith human IgG₁, in Chinese hamster ovary (CHO) cells (Vassar et al.,(1999) Science 286:735-741 and Haniu et al., (2000) J. Biol. Chem.275:21099-21106).

[0127] This construct, referred to as BACE-T-IgG, also contained aprotease cleavage site between BACE and IgG₁, sensitive to humanα-thrombin. The cDNA for the catalytic domain of human BACE (residues1-460) was PCR-amplified and subcloned into the mammalian expressionvector pTV1.6, upstream of a thrombin cleavage site linked to cDNAencoding human IgG₁ heavy chain. The vector construct,pTV1.6-BACE-T-IgG, was used to produce stably transfected DHFR-deficientCHO DG44 cells, which were then scaled up using methotrexate forselection.

[0128] The clarified growth media harvested from CHO DG44 cells whichcontained the fusion protein was loaded onto a rProtein A SEPHAROSE™column (5×20 cm, Amersham Pharmacia Biotech) using a peristaltic pump at4° C., at 4 mL/min. The column was washed with Dulbecco's PBS, pH 7.1, 4mL/min, until baseline absorbance at 280 nm was observed. BACE-T-IgG waseluted from the resin with 0.10 M citrate, pH 3.0, into tubes containing0.5 volumes of 4 M Tris, pH 8. Fractions containing the fusion proteinwere dialyzed extensively using 12,000-14,000 kDa MWCO membrane(UltraPURE, GIBCO BRL) against PBS, pH 7.1, at 4° C. The protein wassterile filtered (0.22 μm) and stored at 4° C.

[0129] To generate BACE for binding experiments, the fusion proteinBACE-T-IgG was treated with human α-thrombin (Enzyme Research Labs,South Bend, Ind.) at a ratio of 1:500 (mass:mass) in Dulbecco's PBS, pH7.1, at 37° C. for 2 hr. Human α-thrombin was removed by passing thesample over Benzamidine SEPHAROSE™ 6B column (Amersham PharmaciaBiotech). The cleaved IgG₁ was captured by passing the solution over arProtein A SEPHAROSE™ (Amersham Pharmacia Biotech) column, whereas theBACE passed through this column. The protein sample was further purifiedby concentrating to 10-15 mg/mL using a centrifugation concentrationunit (Millipore Ultrafree 10 kDa MWCO, 15 mL unit) and loading onto aSUPERDEX™ 200 PC 3.2/30 gel-filtration column (Amersham PharmaciaBiotech). The column was run at 3 mL/min with Dulbecco's PBS, pH 7.1, atroom temperature. Fractions containing BACE were combined, sterilefiltered (0.22 μm) and stored at 4° C. The protein was characterized bySDS-PAGE and other biophysical techniques, including UV-visspectrometry, dynamic and static light scattering, to demonstrate thatit was glycosylated and monomeric. Amino-terminal sequencing indicated amixture of two start sequences, LPRET- and ETDEE-, the latter of whichis the expected sequence for the mature sequence of the protease (Haniuet al., (2000) J. Biol. Chem. 275:21099-21106).

[0130] BACE was prepared for isothermal titrating calorimetry byextensive dialysis against fresh buffer at 4° C. using 12,000-14,000 kDaMWCO dialysis membrane (UltraPURE, GIBCO BRL). The buffer was eitherDulbecco's PBS (2 mM KH₂PO₄, 8 mM Na₂HPO₄, 137 mM NaCl, 3 mM KCl), pH7.1, or 25 mM NaOAc, containing 137 mM NaCl and 3 mM KCl, pH 5.3.Following 2 buffer changes, the protein was removed from the membraneand centrifuged (5 min×4000 g, 4° C.) to remove particulates. Theprotein was stored at 4° C. until needed for the calorimetryexperiments. The protein concentration was determined by 10-folddilution into the same buffer, and measuring the UV absorbance at 280 nmin a 1.00 cm pathlength cell (calculated value=1.22 AU=1.00 mg/mLprotein, based on mean glycosylated molecular weight=53.5 kDa, and givenamino acid composition). Final concentrations used in isothermaltitrating calorimetry were typically 5.0 μM, containing a finalconcentration of 0.5% v/v DMSO.

[0131] Peptides were dissolved in the buffer dialysate from proteindialysis, and equal volumes of DMSO were added to each (0.5% v/v DMSO).The pH values of the solutions were adjusted as necessary to equal thatof the buffer dialysate and protein sample within 0.01 pH units. Theconcentrations of peptides NLTTYPYFIPLP (SEQ ID NO:19) and ALYPYPLPISAK(SEQ ID NO:20) were determined by diluting 10-fold in the same bufferand measuring the UV absorbance at 276 nm, using the value of ε=2780M⁻¹cm⁻¹ (or 2×1390 M⁻¹cm⁻¹ for 2 Tyr residues per peptide).

[0132] The active site inhibitor peptide OM99-2 was obtained from Bachem(King of Prussia, Pa.) as a dry white powder. The compound was weighedinto a clean polypropylene tube (1.7 mL) and DMSO was added to prepare astock solution of 10.0 mM. This stock sample was diluted in buffer(protein dialysate) to −50 μM containing a final concentration of 0.5%v/v DMSO for titration experiments.

[0133] Isothermal titrating calorimetry experiments were performed witha VP-ITC instrument from MicroCal, Inc. (Northampton, Mass.). Theinstrument was controlled with a personal computer, and thermallyregulated at the desired experimental temperature (25° C. or 37° C.).Samples of BACE and peptides were degassed for 2×5 min at 15° C. using atemperature-regulated degassing unit (MicroCal) before loading into thesample chamber or syringe, respectively. Deionized, degassed water wasloaded into the instrument reference chamber and used for allexperiments. For each titration experiment, a fresh sample of BACE(typically 5.0 μM, 2.0 mL) was loaded into the instrument sample chamber(volume=1.438 mL), using a glass syringe, following the manufacturer'sdirections. Similarly, fresh peptide samples (typically ˜150 μM forpeptides NLTTYPYFIPLP (SEQ ID NO:19) and ALYPYPLPISAK (SEQ ID NO:20) and˜50 μM for OM99-2, 0.3 mL total volume) were loaded into the instrumentinjecting syringe unit before each experiment. For experiments todemonstrate that the active site directed inhibitor peptide OM99-2 andthe peptide NLTTYPYFIPLP (SEQ ID NO:19) did not compete for the samesite, a 10-fold excess of desired peptide was first added to a freshsample of BACE and incubated at room temperature for 5 min beforedegassing and loading into the instrument. Titrations were thenperformed with the other peptide in the syringe.

[0134] Instrument Parameters

[0135] The temperature was maintained at 25° C. or 37° C. during thetitration experiments. A power setting of 6.0 μCal/sec was used, and asyringe stirring rate of 300 rpm was used. The initial injection waskept at 1.5-2.0 μL and the data from this injection was not included inthe analysis as a standard practice. To completely define the bindingisotherm, typically a 2.5-fold to 3.5-fold excess of peptide was addedduring the course of the titration experiment, using about 15 injectionsper molar equivalent, or 3.0 μL (NLTTYPYFIPLP, SEQ ID NO:19) or 6.0 μL(OM99-2) per injection. The data collection time per injection was fixedat 360 sec, with a signal averaging time of 2 sec. The data was analyzedusing the manufacturer's software fitting to a single site binding model(i.e. Origin 5.0 for ITC). Before molar heat calculations were done,background corrections were made on all peaks by subtracting the mean ofthe final 10-15 injections from all injections.

[0136] The calculated molar heat values were fitted to a single bindingsite model using the manufacturer's software to determine the bindingstoichiometry (n), the association constant (K_(A)), the enthalpy of thereaction (ΔH), and the entropy of the reaction (ΔS). These values wereused to calculate the dissociation constant (K_(d)) which is thereciprocal of K_(A), and the Gibbs free energy of the reaction (ΔG),which is related to the K_(A), ΔH, and ΔS by the following equations:ΔG=−RT (ln (K_(A)))=ΔH−TΔS (Levine, Physical Chemistry, (2^(nd) ed.),McGraw-Hill Co., (1983), p. 125).

[0137] The sample cell was cleaned between injections by washingextensively with PBS, H₂O, and again with PBS. After multipleexperiments (typically 6-8), the sample cell and syringe were moreextensively cleaned using manufacturer's recommendations with adetergent solution heated to 50° C., followed by extensive washing withH₂O, methanol, H₂O, and finally PBS. Blank injections of buffer intobuffer were then performed to establish sufficient cleaning andreproducible background before carrying out additional BACE-peptideexperiments.

[0138] Results

[0139] Titrations with peptides NLTTYPYFIPLP (SEQ ID NO:19) andALYPYPLPISAK (SEQ ID NO:20) into BACE demonstrated saturable 1:1 bindingin Dulbecco's PBS, pH 7.1 at 25° C. (below, FIGS. 1-2). The bindingconstants were determined to be K_(d)=61 nM for NLTTYPYFIPLP (SEQ IDNO:19) and K_(d)=113 nM for ALYPYPLPISAK (SEQ ID NO:20).

[0140] Further experiments were carried out with BACE at pH 5.3 and 37°C. with NLTTYPYFIPLP (SEQ ID NO:19) to investigate the binding of thispeptide under catalytically active conditions in both the absence andpresence of the active site inhibitor peptide OM99-2. Representativeintegrated data fitted to a single site model are given below in FIG. 3for the experiment in which a 10-fold excess of OM99-2 was first addedto BACE, and the complex was then titrated with NLTTYPYFIPLP (SEQ IDNO:19), at pH 5.3, 37° C. The determined thermodynamic parameters forthe complete sets of experiments are given in Table 3, and allexperiments showed saturable binding and excellent fits to a single sitemodel. TABLE 3 Calculated and Fitted Data for Four Experiments withBACE, OM99-2, and Peptide #1, Conducted at pH 5.3, 37° C. Titrant ΔGT(dS) ΔH K_(d) Stoichiometry Sample Cell Contents (syringe) (kcal/mol)(kcal/mol) (kcal/mol) (nM) (mole:mole) CHO BACE Peptide #1* −9.1 −6.8−15.9 380 0.93:1.00 CHO BACE: OM99-2 Peptide #1 −9.3 −5.7 −15.0 2800.96:1.00 CHO BACE OM99-2 −11.9 −5.3 −17.2 4 1.01:1.00 CHO BACE: Peptide#1 OM99-2 −12.1 −4.0 −16.1 3 1.03:1.00

[0141] These experiments demonstrated that binding of peptideNLTTYPYFIPLP (SEQ ID NO:19), and OM99-2 to BACE were not mutuallyexclusive, and that the binding was not strongly coupled, as shown belowin Scheme 1.

Example 4

[0142] Fluorescently Labeled EBPs Binding to BACE Assay

[0143] An assay to evaluate the binding of BACE to EBPs labeled with afluorescent molecule (for example, but not limited to Alexa488) wasdeveloped. This assay uses the catalytic domain of human BACE expressedin a CHO cell line (according to Example 3) and labeled EBPs such asMolecule X shown in FIG. 4. The change in fluorescent anisotropy of theEBP peptide upon binding to BACE is monitored.

[0144] Peptides were dissolved in 100% DMSO (dimethyl sulfoxide) at 10mM concentration, and then diluted 10-fold into deionized water. Theconcentration of the labeled peptides was determined by their absorbanceat 495 nm (ε=71000 cm⁻¹M⁻¹). The concentration of selected unlabeledpeptides was determined by their Tyr absorbance at 276 nm (ε=1390cm⁻¹M⁻¹ per Tyr residue).

[0145] The binding was carried out at pH 7.1 (PBS buffer) and pH 4.5 (50mM acetate buffer) in the presence of 1% DMSO. Fluorescence anisotropywas measured at 25° C. in an AVIV fluorometer. The excitation andemission wavelengths were set to 495 and 519 nm, respectively. Theexcitation and emission slit width were 4 and 10 nm, respectively. Aconcentrated BACE stock was used to titrate a 300 μl solution of 10 nMlabeled EBP. The final BACE concentration ranges from 10 to 5000 nM.After the addition of BACE, the solution was mixed for 10 times with apipettor. The fluorescence anisotropy was averaged over a 5 minuteperiod.

[0146] The change in anisotropy was plotted against the BACEconcentration (see, for example, FIG. 5). The K_(d) of the labeled EBPas well as the initial (r₀) and final anisotropy (r_(b)) of the labeledEBP were calculated from curve fitting using the program Kaleidagraph.The equation used to fit the binding data is identical to equation 1 inLai et al., (2000) Arch. Biochem. Biophys. 381:278-284.

Example 5

[0147] Competitive Binding Assay

[0148] The competitive binding assay was carried out at pH 7.1 (PBSbuffer) or pH 4.5 (50 mM acetate buffer). The fluorescence anisotropywas measured at 25° C. in an AVIV fluorometer. The excitation andemission wavelengths were set to 495 and 519 nm, respectively. Theexcitation and emission slit widths were 4 and 10 nm, respectively.

[0149] Labeled EBP (Molecule X) at 10 nM was mixed with BACE at aconcentration equal to the K_(d). The initial anisotropy value wasmeasured. A concentrated unlabeled peptide or compound stock wastitrated into the above solution of EBP and BACE. The finalconcentration of unlabeled peptide or compound ranges from 20 to 20000nM. After each addition, the solution was mixed for 10 times with apipettor. The fluorescence anisotropy was averaged over a 5 minuteperiod.

[0150] The change in anisotropy was converted to fractional occupancybased on the r₀ and r_(b) obtained from the binding assay. Thefractional occupancy was then plotted against the concentration of thecompeting peptide or compound. (see, for example, FIG. 6) The K_(d) ofthe competing ligand was calculated from curve fitting using the programKALEIDAGRAPH™ (Synergy Software, Reading, Pa.). The equation used to fitthe competition data is identical to equation 4 in Lai et al., (2000)Arch. Biochem. Biophys. 381:278-284.

Example 6

[0151] Binding of Labeled EBP (Molecule X) to BACE at pH 7.1 and pH 4.5

[0152] Binding of a labeled EBP (Molecule X, FIG. 4) to BACE was carriedout at pH 7.1 and pH 4.5. By fitting of the data to a 1:1 binding model,the binding constants were determined to be 139 and 904 nM at pH 7.1 andpH 4.5, respectively.

Example 7

[0153] Determination of Binding Affinity of Unlabeled EBP using theCompetition Assay

[0154] The binding affinity of peptide NLTTYPYFIPLP (SEQ ID NO:19)(unlabeled Molecule X) was determined using the competition assay asdescribed in Example 6. The binding constants were 84 and 1073 nM at pH7.1 and pH 4.5, respectively. The ability of unlabeled EBP to displaceMolecule X from binding to BACE suggests that the labeled and unlabeledpeptides bind to BACE at the same exosite. The binding constants of thisEBP with and without Alexa488 label at essentially the same at both pHs,indicating that the presence of the Alexa488 label does not affect thebinding interactions of the EBP to BACE. The binding constant at pH 7.1,determined by the fluorescent anisotropy, is consistent with the ITCresult (61 nM) of Example 3. At pHs where BACE will be catalyticallyactive, the binding of the EBP NLTTYPYFIPLP (SEQ ID NO:19) is weaker(1073 nM at pH 4.5 by fluorescence anisotropy and 380 nM at pH 5.3 byITC). Unless otherwise specified, subsequent binding and competitionexperiments were carried out at pH 4.5, the pH optimum of theproteolytic activity of BACE.

Example 8

[0155] Screening of Truncated Peptides to Define the Minimal LengthRequirement for Binding

[0156] A collection of truncated peptides (from N-, from C-, and fromboth N- and C-termini of peptide NLTTYPYFIPLP (SEQ ID NO:19)) wasscreened in the competition assay described above in Example 6 withslight modification. Molecule X was used as the labeled EBP (K_(d)=1.0μM at pH 4.5). The truncated unlabeled peptides were screened at asingle concentration of 10 μM at pH 4.5. The anisotropy values detectedwith Molecule X in the absence of inhibitor, i.e., truncated unlabeledpeptide (r_(a)) and in the presence of the inhibitor (r_(b)) and thelabeled peptide alone (r₀) were used to calculate the percent ofinhibition: 100 (r_(a)-r_(b))/(r_(a)-r₀). The percent of inhibition wascompared among the truncated peptides. It was determined that theN-terminal 4 residues and the C-terminal residue in peptide NLTTYPYFIPLP(SEQ ID NO:19) were not critical in binding (FIG. 7, upper and lowerpanels). Therefore, it was determined that the minimal length desiredfor binding is a 7-mer peptide. A preferred 7-mer BACE exosite bindingpeptide was identified having the sequence of YPYFIPL (SEQ ID NO:10),corresponding to amino acids 5-11 in the original NLTTYPYFIPLP (SEQ IDNO:19) peptide.

[0157] In addition to Molecules X, Yn, and Z, the following BACE exositebinding peptides were identified: Compound Se- Number quence Amino AcidComposition BMS-561871 1-12 NLTTYPYFIPLP (SEQ ID NO:19) BMS-593925 5-11YPYFIPL (SEQ ID NO:10) BMS-590022 2-12 LTTYPYFIPLP (SEQ ID NO:44)BMS-590023 3-12 TTYPYFIPLP (SEQ ID NO:45) BMS-590024 4-12 TYPYFIPLP (SEQID NO:46) BMS-590008 5-12 YPYFIPLP (SEQ ID NO:47) BMS-590014 1-11NLTTYPYFIPL (SEQ ID NO:48) BMS-599191 5-11^(a) YPYFIAL (SEQ ID NO:49)BMS-599192 5-11^(a) YPYFIPA (SEQ ID NO:50) BMS-599195 5-11^(b) YPBFIPL(SEQ ID NO:51) BMS-599199 5-11^(b) YPYFIPB (SEQ ID NO:52) BMS-6076415-11^(d) YPYFIPB-Alexa488 (SEQ ID NO:108) BMS-607649 5-11^(d)YPBFIPL-Alexa488 (SEQ ID NO:109)

Example 9

[0158] Characterization of EBPs Labeled at Different Positions and withLinkers of Different Lengths

[0159] A comparison of EBPs labeled with Alexa488 at different positions(Molecules X, Y1, Z) was performed using the above binding assay. It ispreferred that the labeled EBP exhibit tight binding affinity and a highsignal to background ratio (i.e., ratio of the anisotropy upon bindingto BACE and the anisotropy of the free EBP peptide). The bindingconstants of Molecules X, Y1, and Z were determined to be 903, 58, and6430 nM, respectively. Molecule Y1 not only exhibits the tightestaffinity for BACE, but also the best signal to background ratio. Anexample of Molecule Y1 binding to BACE is shown in FIG. 8.

[0160] Analogs of Molecule Y1 were prepared with different length oflinkers (FIG. 4). The binding constants of Y1, Y2, Y3, and Y4 were 57,92, 48, 62 nM, respectively. The lack of change in the affinity ofMolecules Yn (where n=1-4) for BACE indicates that the length of linkerbetween Alexa488 and the peptide does not affect the strength of bindinginteraction.

Example 10

[0161] Binding of Molecule Y1 can be Displaced by Unlabeled EBP

[0162] Unlabeled EBP corresponding to Molecule Y1 (BMS-593925, YPYFIPL(SEQ ID NO:10)) was used in the competition assay to displace MoleculeY1 from binding to BACE at pH 4.5 (see FIG. 9). The unlabeled EBP wasfound to bind to BACE with a K_(d) of 1197 nM. This demonstrates thatalthough the presence of the C-terminal Alexa488 on Molecule Y1increased the affinity of Y1 for BACE, it still binds to the sameexosite as the unlabeled peptide since it can be displaced by theunlabeled peptide.

Example 11

[0163] Binding of a Labeled EBP to BACE in the Presence of an ActiveSite Inhibitor

[0164] The binding assay described herein above was used to determinethe binding affinity of Molecule Y1 to BACE in the presence of 10 μMOM99-2, a known BACE inhibitor that binds to BACE at the active sitewith a K_(i) of 2 nM (FIG. 10). Before the addition of BACE, aconcentrated stock (1 mM in 100% DMSO) of OM99-2 was mixed with the 300μl solution of 10 nM labeled EBP (molecule Y1) to a final OM99-2concentration of 10 μM. BACE was then titrated into this mixture ofOM99-2 and labeled EBP as described above. It was found the EBP(molecule Y1) binds to BACE with somewhat enhanced affinity (5-fold) inthe presence of 10 μM OM99-2. This demonstrates that the EBP does notbind to BACE at the same site as OM99-2, i.e., it binds at an exositeaway from the active site. The binding of OM99-2 at the active site mayhave a positively cooperative effect on the EBP binding.

Example 12

[0165] Binding of Labeled EBP to BACE Purified from E. coli Cells

[0166] Binding of Molecule Y1 to the catalytic domain of BACE purifiedfrom E. coli cells was carried out to determine the effect ofglycosylation on the EBP binding. It is known that proteins purifiedfrom E. coli do not have glycosylation. The binding affinity of E. coliexpressed human BACE for EBPs was found to be the same as that of humanBACE purified from CHO cells, indicating that the EBPs are indeedbinding to the BACE protein, rather than the sugar groups.

Example 13

[0167] Determination of the Contribution of Each Amino Acid in EBP byAla Scan and Bpa Scan

[0168] A collection of mutated peptides base on peptide YPYFIPL (SEQ IDNO:10) was screened using the competition assay described in Example 9.

[0169] The results of the Ala scan shown in FIG. 11 (upper panel)suggests that while the last two amino acids (PL) are not critical forthe binding interaction, the other five amino acids (YPYFI; SEQ ID NO:2)all play an important role in the interaction of the EBP with BACE.

[0170] The result of the scan of benzophenone-containing peptides (seeFIG. 11, lower panel) suggests that Leu-11 can be replaced with abenzophenone (Bpa) group. Tyr-7 accommodates a Bpa substitution betterthan most other positions, but not as well as Leu-11.

Example 14

[0171] Photo-Crosslinking of Bpa Containing EBPs to BACE

[0172] Two EBPs containing a Bpa substitution as well as an Alexa488group attached at the C-terminus (YPYFIPB-Alexa488 (SEQ ID NO:108) andYPBFIPL-Alexa488 (SEQ ID NO:109); where B indicates a benzophenonegroup) were tested for their binding to BACE. They were both found tobind to BACE reversibly with affinities around 100 nM in the absence ofUV light. Both peptides were used to covalently crosslink to BACE uponUV irradiation at 360 nm. The crosslinking reaction was carried out atvarious temperatures in the presence of 2 μM BACE, various amounts ofEBP containing the Bpa group, as well as 100 μM of a scrambled peptidewith the sequence of LYPPYIF (SEQ ID NO:53) that does not bind to BACE.The reaction mixture was separated by SDS-PAGE and visualized on aFluoroimager (Molecular Dynamics) with an excitation of 488 nm and anemission of 530 nm. FIG. 12 shows a typical time course of thecrosslinking reaction of BMS-607641. The Bpa containing EBPs can becovalently crosslinked to BACE and thus serve as tools for thedetermination of the structure of the exosite on BACE.

Example 15

[0173] Assay for Identifying BACE Exosite Binding Compounds

[0174] Compounds that bind to BACE at the exosite can be discoveredusing the competition assay described in Examples 5 and 9. By mixingcompounds at a single concentration or varying concentrations with afixed concentration mixture of BACE and labeled EBP, changes in thefluorescence anisotropy of the Alexa488 group are followed to determinethe competition (or the lack of) of compounds for the EBP binding toBACE. To facilitate the high throughput discovery of exosite bindingcompounds of BACE, the assay is carried out in a 96, 384, or 1536 wellformat.

Example 16

[0175] Molecule Y3 Inhibits the Proteolytic Activity of BACE

[0176] Molecule Y3 was tested in a BACE cleavage assay using the peptideMCA-EVNLDAEFK(-dnp)-COOH (SEQ ID NO:107) as a substrate. The assay wascarried out essentially as described in Mallender et al., (2001) Mol.Pharmacol. 59:619-626 and in Marcinkeviciene et al., (2001) J. Biol.Chem. 276: 23790-23794. A concentration of 0.1 nM BACE was incubatedwith Molecule Y3 at various concentrations for 15 minutes beforesubstrate peptide was added to a final concentration of 25 μM. Thereaction was allowed to proceed for 60 minutes at 25° C. before it wasstopped by boiling. The reaction mixture was separated on a C18 columnusing reverse phase HPLC (Waters, Milford, Mass.). The IC₅₀ value wascalculated using the Langmuir isotherm equation (Copeland, R. A.,Enzymes: A Practical Introduction to Structure, Mechanism, and DataAnalysis, (2^(nd) ed), Wiley-VCH, New York, N.Y. (2000)).

[0177] Molecule Y3 was found to inhibit the proteolytic activity of BACEwith an IC₅₀ of 731 nM (FIG. 13), demonstrating that binding of EBPs tothe exosite on BACE can indeed interfere with the catalytic activity ofBACE. The inhibition by EBP may be more potent when protein substrates,containing an APP sequence, are used instead of short peptide substrate.

Example 17

[0178] Peptide Synthesis

[0179] The EBP peptides described herein were prepared using either anApplied Biosystems Inc. 433A peptide synthesizer or an Advanced ChemtechMultiple Peptide Synthesizer (MPS-396). The MPS-396 synthesizer was usedto prepare several peptides simultaneously. The ABI 433A synthesizer wasused to prepare individual peptides one at a time.

[0180] The syntheses of the peptide analogs described herein were alsocarried out either by using an Advanced Chemtech Multiple PeptideSynthesizer (MPS-396) or an Applied Biosystems Inc. peptide synthesizer.The step-wise solid phase peptide synthesis was carried out utilizingthe Fmoc/t-butyl protection strategy. The amino acid derivatives usedfor the chain building were protected by the Fmoc group at the α-amino,and the side chain functionalities were protected by groups that areresistant to piperidine treatment, but ultimately cleavable bytrifluoroacetic acid.

Example 18

[0181] Simultaneous Solid Phase Peptide Synthesis of EBP Peptides

[0182] 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methylbenzhydrylamine resin (Rink amide MBHA resin; loading: 0.5 mmol/g) wasloaded as a suspension in dichloromethane/DMF (60:40) into the 96-wellreactor of an Advanced ChemTech MPS 396 synthesizer in volumescorresponding to 0.01-0.025 mmol (20-50 mg) of resin per reactor well.The reactor was placed on the instrument and drained. The wells werethen washed with DMF (0.5-1.0 mL, 3×2 min) and subjected to the numberof automated coupling cycles required to assemble the respective peptidesequences as determined by the pre-programmed sequence synthesis table.The detailed stepwise synthesis protocol used for a typical 0.01mmol/well simultaneous synthesis of 96 compounds is described below.This protocol was adapted for the simultaneous synthesis of arrays ofanalogs. The general synthesis protocol is depicted in Scheme 2.

[0183] Prior to starting the synthesis, the following reagent solutionswere prepared and placed on the instrument as required: 1.5 M (15%)piperidine in DMF; 0.5 M DIEA in NMP; 0.36 M DIC in NMP; 1 M (10%)acetic anhydride in DMF. The required Fmoc-protected amino acids wereprepared as 0.36 M solutions in 0.36 M HOAt/NMP and placed into theappropriate positions in the 32-position amino acid rack.

[0184] Coupling of the amino acid residue was carried out by automatedaddition of a 0.36 M solution of the appropriate Fmoc-amino acid (0.072mmol, 7.2 eq.) and HOAt (7.2 eq.) in NMP (0.2 mL) to all relevant wells.This was followed by addition of a 0.36 M solution of DIC (0.072 mmol,7.2 eq.) in NMP (0.2 nL). The coupling was allowed to proceed for 2 hrs.After reactor draining by nitrogen pressure (3-5 psi) and washing thewells with NMP (1×0.5 mL), the coupling was repeated as described above.At the end of the coupling cycle, the wells were treated with 1M aceticanhydride in DMF (1×0.5 mL, 30 min.) and finally washed with DMF (3×0.5mL).

[0185] An identical coupling protocol was repeated additional times inorder to complete the sequence assembly of the desired peptide analogs.

[0186] Finally, the Fmoc group was removed with 20% piperidine in DMF asdescribed above, and the peptidyl-resins were washed with DMF (4×0.5 mL)and DCM (4×0.5 mL). They were then dried on the reactor block byapplying a constant pressure of nitrogen gas (5 psi) for 10-15 min.

[0187] Cleavage/Deprotection

[0188] The desired peptides were cleaved/deprotected from theirrespective peptidyl-resins by treatment with a TFA cleavage mixture asfollows. A solution of TFA/water/tri-isopropylsilane (94:3:3) (1.0 mL)was added to each well in the reactor block, which was then vortexed for2 hrs. The TFA solutions from the wells were collected by positivepressure into pre-tared vials located in a matching 96-vial block on thebottom of the reactor. The resins in the wells were rinsed twice with anadditional 0.5 mL of TFA cocktail and the rinses were combined with thesolutions in the vials. These were dried in a SpeedVac™ (Savant) toyield the crude peptides, typically in >100% yields (20-40 mgs). Thecrude peptides were either washed with ether or more frequentlyre-dissolved directly in 2 mL of DMSO or 50% aqueous acetic acid forpurification by preparative HPLC as follows.

[0189] Preparative HPLC Purification of the Crude Peptides

[0190] Preparative HPLC was carried out either on a Waters Model 4000 ora Shimadzu Model LC-8A liquid chromatograph. Each solution of crudepeptide was injected into a YMC S5 ODS (20×100 mm) column and elutedusing a linear gradient of MeCN in water, both buffered with 0.1% TFA.The desired product eluted well separated from impurities, typicallyafter 8-10 min., and was collected in a single 10-15 mL fraction on afraction collector. The desired peptides were obtained as amorphouswhite powders by lyophilization of their HPLC fractions.

[0191] HPLC Analysis of the Purified Peptides

[0192] After purification by preparative HPLC as described above, eachpeptide was analyzed by analytical RP-HPLC on a Shimadzu LC-10AD orLC-10AT analytical HPLC system consisting of: a SCL-10A systemcontroller, a SIL-10A auto-injector, a SPD10AV or SPD-M6A UV/VISdetector, or a SPD-M10A diode array detector. A YMC ODS S3 (4.6×50 mm)column was used and elution was performed using a linear gradient ofMeCN in water, both buffered with 0.1% TFA. Mobile phase A: 0.1%TFA/water; mobile phase B: 0.1% TFA/acetonitrile. The purity wastypically >90%.

[0193] Characterization by Mass Spectrometry

[0194] Each peptide was characterized by electrospray mass spectrometry(ES-MS) either in flow injection or LC/MS mode. Finnigan SSQ7000 singlequadrupole mass spectrometers (ThermoFinnigan, San Jose, Calif.) wereused in all analyses in positive and negative ion electrospray mode.Full scan data was acquired over the mass range of 300 to 2200 amu for ascan time of 1.0 second. The quadrupole was operated at unit resolution.For flow injection analyses, the mass spectrometer was interfaced to aWaters 616 HPLC pump (Waters Corp., Milford, Mass.) and equipped with anHTS PAL autosampler (CTC Analytics, Zwingen, Switzerland). Samples wereinjected into a mobile phase containing 50:50 water:acetonitrile with0.1% ammonium hydroxide. The flow rate for the analyses was 0.42 mL/min.and the injection volume 6 μL. A ThermoSeparations Constametric 3500liquid chromatograph (ThermoSeparation Products, San Jose, Calif.) andHTS PAL autosampler were used for LC/MS analyses. Chromatographicseparations were achieved employing a Luna C₁₈, 5 micron column, 2×30 mm(Phenomenex, Torrance, Calif.). The flow rate for the analyses was 1.0mL/min and column effluent was split, so that the flow into theelectrospray interface was 400 μL/min. A linear gradient from 0% to 100%B in A over 4 minutes was run, where mobile phase A was 98:2water:acetonitrile with 10 mM ammonium acetate and mobile phase B was10:90 water:acetonitrile with 10 mM ammonium acetate. The UV responsewas monitored at 220 nm. The samples were dissolved in 200 μL 50:50H₂O:MeCN (0.05% TFA). The injection volume was 5 μl.

[0195] In all cases, the experimentally measured molecular weight waswithin 0.5 Daltons of the calculated mono-isotopic molecular weight.

Example 19

[0196] Solid Phase Synthesis of EBP Peptide Analogs Using an AppliedBiosystems Model 431A Peptide Synthesizer

[0197] Following is the general description for the solid phasesynthesis of typical EBP peptide analogs, using an upgraded AppliedBiosystems Model 433A peptide synthesizer. The upgraded hardware andsoftware of the synthesizer enabled conductivity monitoring of the Fmocdeprotection step with feedback control of coupling. The protocolsallowed a range of synthesis scale from 0.05 to 0.25 mmol.

[0198] 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methylbenzhydrylamine resin (Rink amide MBHA resin; loading: 0.5 mmol/g) (0.1mmol) was placed into a vessel of appropriate size on the instrument,washed 6 times with NMP and deprotected using two treatments with 22%piperidine/NMP (2 and 8 min. each). One or two additional monitoreddeprotection steps were performed until the conditions of the monitoringoption were satisfied (<10% difference between the last twoconductivity-based deprotection peaks). The total deprotection time was10-12 min. The first Fmoc-protected amino acid was coupled next usingthe following method: Fmoc-AA-OH (1 mmol, 10 eq.) was dissolved in 2 mLof NMP and activated by subsequent addition of 0.45 M HBTU/HOBt in DMF(2.2 mL) and 2 M DIEA/NMP (1 mL). The solution of the activatedFmoc-protected amino acid was then transferred to the reaction vesseland the coupling was allowed to proceed for 30 to 60 min., depending onthe feedback from the deprotection steps. The resin was then washed 6times with NMP, and subjected to the additional deprotection/couplingcycles as described above necessary to complete the assembly of thedesired sequence. Finally, the Fmoc group was removed with 22%piperidine in NMP as described above, and the peptidyl-resin was washed6 times with NMP and DCM, and dried in vacuo.

[0199] Cleavage/Deprotection

[0200] The desired peptide was cleaved/deprotected from its respectivepeptidyl-resin by treatment with a solution ofTFA/water/tri-isopropylsilane (94:3:3) (5.0 mL/g of peptidyl-resin) for2 hrs. The resin was filtered off, rinsed with TFA cleavage solution (2mL), and the combined TFA filtrates were dried in vacuo. The resultingsolid was triturated and washed with diethyl ether, and finally dried,to yield the crude peptide product as a white solid. This was purifiedby preparative HPLC as described herein. The fraction containing a pureproduct was lyophilized, to yield the pure peptide product in 20-40%isolated yield.

Example 20

[0201] Coupling of the Alexa488 Label to a EBP Peptide

[0202] The Alexa488 label was attached to either the α-amino group ofthe N-terminal amino acid residue of a EBP peptide or the ω-amino groupof the side chain of a α,ω-diamino acid appended to the C-terminus of aEBP peptide by reaction of the purified EBP peptide with theN-hydroxysuccinimidyl ester of the Alexa Fluor® 488 fluorophore [1.5-2.0eq.] for 16-20 hrs in NMP and DIEA (1-2 eq). The reaction progress wasmonitored by HPLC. The resulting Alexa488-labeled EBP peptide was thenpurified by HPLC and characterized as described herein.

Example 21

[0203] Biased Library Peptides Identified by Solution Panning at pH 5.2

[0204] Panning was performed at pH 5.2 to identify peptides that bind tothe exosite under these conditions more tightly than was the case forthe peptides derived from the unbiased libraries. The methods employedare identical to those described in Example 2, with the exception thatProtein A cells were replaced by Protein A agarose beads (Sigma, St.Louis, Mo.) and that amounts of BACE, number of washes and temperaturesof wash buffers were used as outlined below to maximize recovery of thetightest binding phage. More particularly, two biased M13-based peptidelibraries were panned against BACE-Ig prepared from CHO cells, describedherein above. Protein A beads were preblocked in 50 mM NaOAc, pH 5.2,+2% BSA for 2 hours. In parallel, BACE was incubated for two hours withlibrary phage using the same buffer. Both samples were then mixedtogether for two hours. This step was followed by several washes andphage were eluted with 6M urea, pH 3.0, and used for amplification andfurther panning cycles or DNA sequencing after round three. Cycle 1:10micrograms of BACE were used and 6 quick washes were carried out withPBS plus 0.2% Tween 20 at room temperature. Cycle 2: 50 nanograms ofBACE were used and there were 7 washes of 3 minutes duration each with50 mM NaOAc, pH 5.2, +0.2% Tween 20 at 37° C. Cycle 3: 25 nanograms ofBACE were used and there were 15 washes of three minutes duration eachusing 0.3M NaOAc, pH 5.2, at 37° C.

[0205] Biased peptide libraries were employed in this Example. Thebiased libraries were made as described herein (see also, Sidhu et al.,(2000) Method Enzymol. 328:333-363), except that the residues definingthe core motif (i.e., HYPYFI (SEQ ID NO:54) were fixed in order to biasthe peptides. Each X corresponds to one random library residue. Allpeptides in the table below are preferably synthesized with an addedunblocked N-terminal Ala, while C-termini are preferably blocked. Thelibraries were designed as follows: linear    XXXXXHYPYFIXXXXX (SEQ IDNO:55) library 1: cyclic CysXXXXXHYPYFIXXXXXCys (SEQ ID NO:56) library2:

[0206] Peptides observed to bind to the BACE exosite at pH 5.2 are shownin the table presented below. In the table, the fixed motif is indicatedby italics. Peptides are grouped into sets with shared sequencesimilarity within the random segments and those similarities are inbold. Potential disulfide bonds are indicated by underlining. 6 peptidesgave the most improved phage ELISA binding signal relative to BMS-561871on phage and are identified in the table by “XXX”. The peptides markedXXX are all cyclic, one of which has a third internal Cys. Although itis not the inventors' desire to be bound to any theory of operation, itis noted that the peptide with the internal Cys may be interesting in ascenario in which the peptides bind through the fixed core motif andthen sterically interfere with the access of substrate to the activesite. In this case, it may be that this peptide, and others like it, arebetter inhibitors compared to other peptides that exhibit the sameaffinity. TABLE 4 Exosite Binding Peptides Identified by SolutionPanning at pH 5.2  TDQPK HYPYFI PSPHS SEQ ID NO:58  THQPK HYPYFI PYHHDSEQ ID NO:59  MDHEK HYPYFIEYKHV SEQ ID NO:60 CTEANK HYPYFI PRHSSC SEQ IDNO:61  HSLAPHYPYFIDLHST SEQ ID NO:62  GSQALHYPYFIPYHKH SEQ ID NO:63------------------------------------- CTNKHD HYPYFIRPGEFC SEQ ID NO:64CENKHD HYPYFISAGNYC SEQ ID NO:65 CQTKVMHYPYFIREGVTC SEQ ID NO:66CGPKHLHYPYFISATSRC SEQ ID NO:67 XXX CAAKHSHYPYFIPACSSC SEQ ID NO:68------------------------------------- CASTYPHYPYFIAT CKTC SEQ ID NO:69CAEAKQHYPYFIKW CKTC SEQ ID NO:70 CAEAKGHYPYFI CTTGNC SEQ ID NO:71------------------------------------- CAQAREHYPYFIDLRTV SEQ ID NO:72CAKAPRHYPYFISAQNAW SEQ ID NO:73 CAKASHHYPYFI NLANNG SEQ ID NO:74 CARAITHYPYFI PYCEEC SEQ ID NO:75 XXX  AVSQT HYPYFI PLSQA SEQ ID 110:76------------------------------------- CEDRPTHYPYFI SLNKQC SEQ ID NO:77CKTQDNHYPYFI SLKKAC SEQ ID NO:78 CQTKHQHYPYFI SLTDAC SEQ ID NO:79 XXXCTKAHT HYPYFISNSKIC SEQ ID NO:80 CHHKHT HYPYFI PNTKSC SEQ ID NO:81CSQHHTHYPYFI PSNGMC SEQ ID NO:82 XXX------------------------------------- CAVEAR HYPYFI NTCSNC SEQ ID NO:83CSVVNR HYPYFI NNSSKC SEQ ID NO:84 CTGCAR HYPYFIEVSTQW SEQ ID NO:85------------------------------------- CSNASHHYPYFI STHSTC SEQ ID NO:86CSNPTGHYPYFI SPQGTC SEQ ID NO:87 CNSTPRHYPYFI SVNSTC SEQ ID NO:88CGVQLVHYPYFLPANSTC SEQ ID NO:89 -------------------------------------CARTPSHYPYFISLPDRG SEQ ID NO:90 CSAGHNHYPYFITLPGYG SEQ ID NO:91CASQDYHYPYFI PSPAWG SEQ ID NO:92  ELPFQHYPYFIDLPPV SEQ ID NO:93 MHPNPHYPYFI PLPTR SEQ ID NO:94 CDSCVTHYPYFINTPYKY SEQ ID NO:95 CAKPKQHYPYFI CYPHEC SEQ ID NO:96  INKTQ HYPYFIEYPFH SEQ ID NO:97------------------------------------- CPNTQH HYPYFIKNGEHC SEQ ID NO:98XXX CPDIAH HYPYFIDSKSHC SEQ ID NO:99 CQPTRH HYPYFIDVTGRC SEQ ID NO:100CQNNHH HYPYFITPTHVC SEQ ID NO:101 CTTTHEHYPYFI DPREAC SEQ ID NO:102 XXXCTTPSRHYPYFI DQLGHC SEQ ID NO:103 CNANHTHYPYFIDISRKC SEQ ID NO:104 QFTHKHYPYFI NISPG SEQ ID NO:105 CNMPHSHYPYFI NPHQSC SEQ ID NO:106

Example 22

[0207] BACE Exosite Binding Studies of Peptide BMS-655507 BACE ProteinPreparation

[0208] BACE samples were prepared for isothermal titration calorimetryby extensive dialysis against freshly prepared buffer at 4° C. using12,000-14,000 kDa MWCO dialysis membrane (UltraPURE, GIBCO BRL). Thebuffers used for these experiments were either Dulbecco's PBS (2 mMKH₂PO₄, 8 mM Na₂HPO₄, 137 mM NaCl, 3 mM KCl), pH 7.0, or 50 mM NaOAc, pH4.5. The pH values were determined at room temperature. Following twochanges of buffer (500 mL each), the protein (typically 2-3 mL) wasremoved from the membrane and centrifuged (5 min×4000 g, 4° C.) toremove particulates. Following dialysis, the dialysate was filtered(0.22 μm) and retained for preparation of the peptide samples (below)and rinsing the sample cell of the calorimeter between experiments. Theprotein was stored at 4° C. until needed for the calorimetryexperiments. The protein concentration was determined by 10-folddilution into the same buffer, and measuring the UV absorbance at 280 nmin a 1.00 cm pathlength cell (calculated value=1.22 au=1.00 mg/mLprotein, based on mean glycosylated molecular weight=53.5 kDa, and givenamino acid composition). Final concentrations used in isothermaltitration calorimetry were typically 4-5 μM, containing a finalconcentration of 1% v/v DMSO.

[0209] Preparation of Peptide BMS-655507

[0210] The peptide BMS-655507 (Ac-His-Trp-Pro-Phe-Phe-Ile-Arg-Ser; SEQID NO:57) was dissolved in the buffer dialysate, and a volume of DMSOadded to yield 1.0% v/v. The pH of the peptide solutions were adjustedas necessary to equal that of the buffer dialysate and protein sample(within 0.01 pH units). The concentrations of peptide were determinedmeasuring the UV absorbance at 280 nm, using the molar extinctioncoefficient value of ε=5630 M⁻¹cm⁻¹.

[0211] Isothermal Titration Calorimetry

[0212] Isothermal titration calorimetry experiments were performed witha VP-ITC instrument from MicroCal Inc. (Northampton, Mass.). Theinstrument was controlled with a personal computer, and thermallyregulated at the desired experimental temperature (25° C.). Samples ofBACE and peptides were degassed for 15 min at room temperature using adegassing unit (MicroCal) before loading into the sample chamber orsyringe, respectively. Deionized, degassed water was loaded into theinstrument reference chamber and used for all experiments. For eachtitration experiment, a fresh sample of BACE (typically 5 μM, 2.0 mL)was loaded into the instrument sample chamber (volume=1.438 mL), using aglass syringe, following the manufacturer's directions. Similarly, freshpeptide samples (typically 130-180 μM for BMS-655507, 0.3 mL totalvolume) were loaded into the instrument injecting syringe unit beforeeach experiment.

[0213] Instrument Parameters

[0214] The temperature was maintained at 25° C. during the titrationexperiments. A power setting of 6.0 μCal/sec was used, and a syringestirring rate of 300 rpm was used. The initial injection was kept at1.5-2.0 μL and the data from this injection was not included in theanalysis as a standard practice. To completely define the bindingisotherm, typically a 3-fold to 4-fold excess of peptide was addedduring the course of the titration experiment, using ˜8 injections permolar equivalent, or 4-7 μL (BMS-655507) per injection. The datacollection time per injection was fixed at 360 sec, with a signalaveraging time of 2 sec. The data was analyzed using the manufacturer'ssoftware (i.e. Origin 5.0 for ITC). Before molar heat calculations weredone, background corrections were made on all peaks by subtracting themean of the final 8-12 injections from all injections.

[0215] The calculated molar heat values were fitted to a single bindingsite model using the manufacturer's software to determine the bindingstoichiometry (n), the association constant (K_(A)), the enthalpy of thereaction (ΔH), and the entropy of the reaction (ΔS). These values wereused to calculate the dissociation constant (K_(d)) which is thereciprocal of K_(A), and the Gibbs free energy of the reaction (ΔG),which is related to the K_(A), ΔH, and ΔS by the following equations:ΔG=−RT (In (K_(A)))=ΔH−TΔS (Levine, Physical Chemistry, (2^(nd) ed.),McGraw-Hill Co. (1983), p. 125).

[0216] The sample cell was cleaned between injections by washingextensively with filtered PBS or NaOAc buffer, H₂O, and again withfiltered buffer. After multiple experiments, the sample cell and syringewere more extensively cleaned using manufacturer's recommendations witha detergent solution heated to 50° C., followed by extensive washingwith H₂O, methanol, H₂O, and finally filtered buffer. Blank injectionsof buffer into buffer were then performed to establish sufficientcleaning and reproducible background before carrying out additionalBACE-peptide experiments.

[0217] Results

[0218] Similar to previous experiments with BMS-561871 and BMS-561877,titrations of BMS-655507 into a solution containing BACE demonstratedsaturable 1:1 binding. Experiments were completed in both Dulbecco'sPBS, pH 7.0 and 50 mM NaOAc, pH 4.5, at 25° C. (FIGS. 14 and 15). Thebinding constants were determined to be K_(d)=0.914 μM for at pH 7.0 andK_(d)=1.64 μM at pH pH 4.5, as summarized in the following table. TABLE5 Calculated and Fitted Thermodynamic Data for Experiments with BACE andBMS-655507 Conducted at pH 4.5 And 7.0, 25° C. pH ΔG ΔH K_(d) value(kcal/mol) (kcal/mol) TΔS (kcal/mol) (μM) Stoichiometry pH 4.5 −7.90−13.35 −5.46 1.64 0.86 pH 7.0 −8.23 −18.04 −9.81 0.914 0.71

[0219] Various publications are cited herein that are herebyincorporated by reference in their entirety.

[0220] As will be apparent to those skilled in the art to which theinvention pertains, the present invention may be embodied in forms otherthan those specifically disclosed above without departing from the scopeand spirit of the invention.

1 109 1 4 PRT Artificial Synthesized Peptide 1 Tyr Pro Tyr Phe 1 2 5 PRTArtificial Synthesized Peptide 2 Tyr Pro Tyr Phe Ile 1 5 3 5 PRTArtificial Synthesized Peptide 3 Xaa Tyr Pro Tyr Phe 1 5 4 6 PRTArtificial Synthesized Peptide 4 Xaa Tyr Pro Tyr Phe Xaa 1 5 5 7 PRTArtificial Synthesized Peptide 5 Xaa Tyr Pro Tyr Phe Xaa Xaa 1 5 6 5 PRTArtificial Synthesized Peptide 6 Tyr Pro Tyr Phe Xaa 1 5 7 6 PRTArtificial Synthesized Peptide 7 Tyr Pro Tyr Phe Xaa Xaa 1 5 8 5 PRTArtificial Synthesized Peptide 8 His Tyr Pro Tyr Phe 1 5 9 6 PRTArtificial Synthesized Peptide 9 Tyr Pro Tyr Phe Ile Pro 1 5 10 7 PRTArtificial Synthesized Peptide 10 Tyr Pro Tyr Phe Ile Pro Leu 1 5 11 7PRT Artificial Synthesized Peptide 11 Tyr Pro Tyr Phe Leu Pro Ile 1 5 127 PRT Artificial Synthesized Peptide 12 Tyr Pro Tyr Phe Xaa Pro Ile 1 513 7 PRT Artificial Synthesized Peptide 13 Tyr Pro Tyr Phe Xaa Pro Xaa 15 14 7 PRT Artificial Synthesized Peptide 14 His Tyr Pro Tyr Phe Ile Pro1 5 15 5 PRT Artificial Synthesized Peptide 15 Tyr Pro Tyr Phe Leu 1 516 6 PRT Artificial Synthesized Peptide 16 Tyr Pro Tyr Phe Leu Pro 1 517 7 PRT Artificial Synthesized Peptide 17 His Tyr Pro Tyr Phe Leu Pro 15 18 8 PRT Artificial Synthesized Peptide 18 His Tyr Pro Tyr Phe Ile ProLeu 1 5 19 12 PRT Artificial Synthesized Peptide 19 Asn Leu Thr Thr TyrPro Tyr Phe Ile Pro Leu Pro 1 5 10 20 12 PRT Artificial SynthesizedPeptide 20 Ala Leu Tyr Pro Tyr Phe Leu Pro Ile Ser Ala Lys 1 5 10 21 5PRT Artificial Synthesized Peptide 21 Trp Pro Xaa Phe Ile 1 5 22 23 PRTArtificial Synthesized Peptide 22 Glu Thr Trp Pro Arg Phe Ile Pro TyrHis Ala Leu Thr Gln Gln Thr 1 5 10 15 Leu Lys His Gln Gln His Thr 20 2327 PRT Artificial Synthesized Peptide 23 Thr Ala Glu Tyr Glu Ser Arg ThrAla Arg Thr Ala Pro Pro Ala Pro 1 5 10 15 Thr Gln His Trp Pro Phe PheIle Arg Ser Thr 20 25 24 12 PRT Artificial Synthesized Peptide 24 GlnAsn His Tyr Pro Tyr Phe Ile Ala Val Pro Ile 1 5 10 25 12 PRT ArtificialSynthesized Peptide 25 Glu Gly Asn Lys His Tyr Pro Tyr Phe Ile Lys Val 15 10 26 12 PRT Artificial Synthesized Peptide 26 Thr His Ser His Tyr ProTyr Phe Ile Glu Leu Glu 1 5 10 27 12 PRT Artificial Synthesized Peptide27 Gln Gln Tyr Pro Tyr Phe Ile Pro Val Ile Arg Pro 1 5 10 28 12 PRTArtificial Synthesized Peptide 28 His Tyr Pro Tyr Phe Leu Pro Leu HisThr Pro Lys 1 5 10 29 24 PRT Artificial Synthesized Peptide 29 Ala MetLeu Asp Gly Ala Pro Thr Asn Arg Asn Ser Gln His Tyr Pro 1 5 10 15 TyrPhe Leu Pro Ile Ala Thr Val 20 30 23 PRT Artificial Synthesized Peptide30 Leu Pro Val Tyr Asp Thr Thr Ala Pro Thr His Tyr Pro Tyr Phe Leu 1 510 15 Pro Leu Pro Arg Ile Ser Pro 20 31 12 PRT Artificial SynthesizedPeptide 31 Ser Gln Leu Gln His Tyr Pro Tyr Phe Arg Pro Leu 1 5 10 32 12PRT Artificial Synthesized Peptide 32 Tyr Ile Pro His Tyr Pro Tyr PheIle Arg Leu Asn 1 5 10 33 23 PRT Artificial Synthesized Peptide 33 LysMet His Ser Met Ile Asn Gln Leu Gly Thr Arg His Tyr Pro Tyr 1 5 10 15Phe Arg Glu Ile Asn Asp Tyr 20 34 12 PRT Artificial Synthesized Peptide34 Gly Ser Thr Lys Ser Tyr Pro Tyr Phe Ile His Thr 1 5 10 35 23 PRTArtificial Synthesized Peptide 35 Asp Ile Trp Asn Gly Ala Lys Ala ProLys Asn Ser Met Tyr Pro Tyr 1 5 10 15 Phe Ile Pro Ser Ser Leu Lys 20 3627 PRT Artificial Synthesized Peptide 36 Ile Ser Val Ile Asn Gln Pro AlaGln Asn Met His Pro Arg Gln Met 1 5 10 15 Thr Ala Tyr Pro Tyr Phe ArgPro Ile Ser Arg 20 25 37 23 PRT Artificial Synthesized Peptide 37 AspVal Tyr Pro Tyr Phe Val Ser Ser Asn Glu Gly His Ser Ile Arg 1 5 10 15His Lys Gly Asn Asn Ser Leu 20 38 33 PRT Artificial Synthesized Peptide38 Tyr Pro Tyr Phe Ile Asp Ser His Pro Pro Lys Glu Leu Met Pro His 1 510 15 Ser Trp Val Gln Ser Lys Tyr Pro Ala Ser Pro Gln Thr His Thr Thr 2025 30 Tyr 39 12 PRT Artificial Synthesized Peptide 39 Gly Tyr Pro TyrPhe Leu Asn Leu Lys Asn Ser His 1 5 10 40 12 PRT Artificial SynthesizedPeptide 40 Asn Ser Tyr Pro Tyr Phe Ile His Leu Ser Asn Pro 1 5 10 41 12PRT Artificial Synthesized Peptide 41 His Asp Tyr Pro Tyr Phe Met MetLeu Thr Gly His 1 5 10 42 12 PRT Artificial Synthesized Peptide 42 GlnIle Glu Thr Tyr Pro Tyr Phe Leu Pro Ile Leu 1 5 10 43 12 PRT ArtificialSynthesized Peptide 43 Tyr Tyr Pro Tyr Phe Ile Ser Thr Ala Arg Glu Val 15 10 44 11 PRT Artificial Synthesized Peptide 44 Leu Thr Thr Tyr Pro TyrPhe Ile Pro Leu Pro 1 5 10 45 10 PRT Artificial Synthesized Peptide 45Thr Thr Tyr Pro Tyr Phe Ile Pro Leu Pro 1 5 10 46 9 PRT ArtificialSynthesized Peptide 46 Thr Tyr Pro Tyr Phe Ile Pro Leu Pro 1 5 47 8 PRTArtificial Synthesized Peptide 47 Tyr Pro Tyr Phe Ile Pro Leu Pro 1 5 4811 PRT Artificial Synthesized Peptide 48 Asn Leu Thr Thr Tyr Pro Tyr PheIle Pro Leu 1 5 10 49 7 PRT Artificial Synthesized Peptide 49 Tyr ProTyr Phe Ile Ala Leu 1 5 50 7 PRT Artificial Synthesized Peptide 50 TyrPro Tyr Phe Ile Pro Ala 1 5 51 6 PRT Artificial Synthesized Peptide 51Tyr Pro Phe Ile Pro Leu 1 5 52 6 PRT Artificial Synthesized Peptide 52Tyr Pro Tyr Phe Ile Pro 1 5 53 7 PRT Artificial Synthesized Peptide 53Leu Tyr Pro Pro Tyr Ile Phe 1 5 54 6 PRT Artificial motif 54 His Tyr ProTyr Phe Ile 1 5 55 16 PRT Artificial Synthesized Peptide 55 Xaa Xaa XaaXaa Xaa His Tyr Pro Tyr Phe Ile Xaa Xaa Xaa Xaa Xaa 1 5 10 15 56 18 PRTArtificial Synthesized Peptide 56 Cys Xaa Xaa Xaa Xaa Xaa His Tyr ProTyr Phe Ile Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys 57 8 PRT ArtificialSynthesized Peptide 57 His Trp Pro Phe Phe Ile Arg Ser 1 5 58 16 PRTArtificial Synthesized Peptide 58 Thr Asp Gln Pro Lys His Tyr Pro TyrPhe Ile Pro Ser Pro His Ser 1 5 10 15 59 16 PRT Artificial SynthesizedPeptide 59 Thr His Gln Pro Lys His Tyr Pro Tyr Phe Ile Pro Tyr His HisAsp 1 5 10 15 60 16 PRT Artificial Synthesized Peptide 60 Met Asp HisGlu Lys His Tyr Pro Tyr Phe Ile Glu Tyr Lys His Val 1 5 10 15 61 18 PRTArtificial Synthesized Peptide 61 Cys Thr Glu Ala Asn Lys His Tyr ProTyr Phe Ile Pro Arg His Ser 1 5 10 15 Ser Cys 62 16 PRT ArtificialSynthesized Peptide 62 His Ser Leu Ala Pro His Tyr Pro Tyr Phe Ile AspLeu His Ser Thr 1 5 10 15 63 16 PRT Artificial Synthesized Peptide 63Gly Ser Gln Ala Leu His Tyr Pro Tyr Phe Ile Pro Tyr His Lys His 1 5 1015 64 18 PRT Artificial Synthesized Peptide 64 Cys Thr Asn Lys His AspHis Tyr Pro Tyr Phe Ile Arg Pro Gly Glu 1 5 10 15 Phe Cys 65 18 PRTArtificial Synthesized Peptide 65 Cys Glu Asn Lys His Asp His Tyr ProTyr Phe Ile Ser Ala Gly Asn 1 5 10 15 Tyr Cys 66 18 PRT ArtificialSynthesized Peptide 66 Cys Gln Thr Lys Val Met His Tyr Pro Tyr Phe IleArg Glu Gly Val 1 5 10 15 Thr Cys 67 18 PRT Artificial SynthesizedPeptide 67 Cys Gly Pro Lys His Leu His Tyr Pro Tyr Phe Ile Ser Ala ThrSer 1 5 10 15 Arg Cys 68 18 PRT Artificial Synthesized Peptide 68 CysAla Ala Lys His Ser His Tyr Pro Tyr Phe Ile Pro Ala Cys Ser 1 5 10 15Ser Cys 69 18 PRT Artificial Synthesized Peptide 69 Cys Ala Ser Thr TyrPro His Tyr Pro Tyr Phe Ile Ala Thr Cys Lys 1 5 10 15 Thr Cys 70 18 PRTArtificial Synthesized Peptide 70 Cys Ala Glu Ala Lys Gln His Tyr ProTyr Phe Ile Lys Trp Cys Lys 1 5 10 15 Thr Cys 71 18 PRT ArtificialSynthesized Peptide 71 Cys Ala Glu Ala Lys Gly His Tyr Pro Tyr Phe IleCys Thr Thr Gly 1 5 10 15 Asn Cys 72 17 PRT Artificial SynthesizedPeptide 72 Cys Ala Gln Ala Arg Glu His Tyr Pro Tyr Phe Ile Asp Leu ArgThr 1 5 10 15 Val 73 18 PRT Artificial Synthesized Peptide 73 Cys AlaLys Ala Pro Arg His Tyr Pro Tyr Phe Ile Ser Ala Gln Asn 1 5 10 15 AlaTrp 74 18 PRT Artificial Synthesized Peptide 74 Cys Ala Lys Ala Ser HisHis Tyr Pro Tyr Phe Ile Asn Leu Ala Asn 1 5 10 15 Asn Gly 75 18 PRTArtificial Synthesized Peptide 75 Cys Ala Arg Ala Ile Thr His Tyr ProTyr Phe Ile Pro Tyr Cys Glu 1 5 10 15 Glu Cys 76 16 PRT ArtificialSynthesized Peptide 76 Ala Val Ser Gln Thr His Tyr Pro Tyr Phe Ile ProLeu Ser Gln Ala 1 5 10 15 77 18 PRT Artificial Synthesized Peptide 77Cys Glu Asp Arg Pro Thr His Tyr Pro Tyr Phe Ile Ser Leu Asn Lys 1 5 1015 Gln Cys 78 18 PRT Artificial Synthesized Peptide 78 Cys Lys Thr GlnAsp Asn His Tyr Pro Tyr Phe Ile Ser Leu Lys Lys 1 5 10 15 Ala Cys 79 18PRT Artificial Synthesized Peptide 79 Cys Gln Thr Lys His Gln His TyrPro Tyr Phe Ile Ser Leu Thr Asp 1 5 10 15 Ala Cys 80 18 PRT ArtificialSynthesized Peptide 80 Cys Thr Lys Ala His Thr His Tyr Pro Tyr Phe IleSer Asn Ser Lys 1 5 10 15 Ile Cys 81 18 PRT Artificial SynthesizedPeptide 81 Cys His His Lys His Thr His Tyr Pro Tyr Phe Ile Pro Asn ThrLys 1 5 10 15 Ser Cys 82 18 PRT Artificial Synthesized Peptide 82 CysSer Gln His His Thr His Tyr Pro Tyr Phe Ile Pro Ser Asn Gly 1 5 10 15Met Cys 83 18 PRT Artificial Synthesized Peptide 83 Cys Ala Val Glu AlaArg His Tyr Pro Tyr Phe Ile Asn Thr Cys Ser 1 5 10 15 Asn Cys 84 18 PRTArtificial Synthesized Peptide 84 Cys Ser Val Val Asn Arg His Tyr ProTyr Phe Ile Asn Asn Ser Ser 1 5 10 15 Lys Cys 85 18 PRT ArtificialSynthesized Peptide 85 Cys Thr Gly Cys Ala Arg His Tyr Pro Tyr Phe IleGlu Val Ser Thr 1 5 10 15 Gln Trp 86 18 PRT Artificial SynthesizedPeptide 86 Cys Ser Asn Ala Ser His His Tyr Pro Tyr Phe Ile Ser Thr HisSer 1 5 10 15 Thr Cys 87 18 PRT Artificial Synthesized Peptide 87 CysSer Asn Pro Thr Gly His Tyr Pro Tyr Phe Ile Ser Pro Gln Gly 1 5 10 15Thr Cys 88 18 PRT Artificial Synthesized Peptide 88 Cys Asn Ser Thr ProArg His Tyr Pro Tyr Phe Ile Ser Val Asn Ser 1 5 10 15 Thr Cys 89 18 PRTArtificial Synthesized Peptide 89 Cys Gly Val Gln Leu Val His Tyr ProTyr Phe Leu Pro Ala Asn Ser 1 5 10 15 Thr Cys 90 18 PRT ArtificialSynthesized Peptide 90 Cys Ala Arg Thr Pro Ser His Tyr Pro Tyr Phe IleSer Leu Pro Asp 1 5 10 15 Arg Gly 91 18 PRT Artificial SynthesizedPeptide 91 Cys Ser Ala Gly His Asn His Tyr Pro Tyr Phe Ile Thr Leu ProGly 1 5 10 15 Tyr Gly 92 18 PRT Artificial Synthesized Peptide 92 CysAla Ser Gln Asp Tyr His Tyr Pro Tyr Phe Ile Pro Ser Pro Ala 1 5 10 15Trp Gly 93 16 PRT Artificial Synthesized Peptide 93 Glu Leu Pro Phe GlnHis Tyr Pro Tyr Phe Ile Asp Leu Pro Pro Val 1 5 10 15 94 16 PRTArtificial Synthesized Peptide 94 Met His Pro Asn Pro His Tyr Pro TyrPhe Ile Pro Leu Pro Thr Arg 1 5 10 15 95 18 PRT Artificial SynthesizedPeptide 95 Cys Asp Ser Cys Val Thr His Tyr Pro Tyr Phe Ile Asn Thr ProTyr 1 5 10 15 Lys Tyr 96 18 PRT Artificial Synthesized Peptide 96 CysAla Lys Pro Lys Gln His Tyr Pro Tyr Phe Ile Cys Tyr Pro His 1 5 10 15Glu Cys 97 16 PRT Artificial Synthesized Peptide 97 Ile Asn Lys Thr GlnHis Tyr Pro Tyr Phe Ile Glu Tyr Pro Phe His 1 5 10 15 98 18 PRTArtificial Synthesized Peptide 98 Cys Pro Asn Thr Gln His His Tyr ProTyr Phe Ile Lys Val Gly Glu 1 5 10 15 His Cys 99 18 PRT ArtificialSynthesized Peptide 99 Cys Pro Asp Ile Ala His His Tyr Pro Tyr Phe IleAsp Ser Lys Ser 1 5 10 15 His Cys 100 18 PRT Artificial SynthesizedPeptide 100 Cys Gln Pro Thr Arg His His Tyr Pro Tyr Phe Ile Asp Val ThrGly 1 5 10 15 Arg Cys 101 18 PRT Artificial Synthesized Peptide 101 CysGln Asn Asn His His His Tyr Pro Tyr Phe Ile Thr Pro Thr His 1 5 10 15Val Cys 102 18 PRT Artificial Synthesized Peptide 102 Cys Thr Thr ThrHis Glu His Tyr Pro Tyr Phe Ile Asp Pro Arg Glu 1 5 10 15 Ala Cys 103 18PRT Artificial Synthesized Peptide 103 Cys Thr Thr Pro Ser Arg His TyrPro Tyr Phe Ile Asp Gln Leu Gly 1 5 10 15 His Cys 104 18 PRT ArtificialSynthesized Peptide 104 Cys Asn Ala Asn His Thr His Tyr Pro Tyr Phe IleAsp Ile Ser Arg 1 5 10 15 Lys Cys 105 16 PRT Artificial SynthesizedPeptide 105 Gln Phe Thr His Lys His Tyr Pro Tyr Phe Ile Asn Ile Ser ProGly 1 5 10 15 106 18 PRT Artificial Synthesized Peptide 106 Cys Asn MetPro His Ser His Tyr Pro Tyr Phe Ile Asn Pro His Gln 1 5 10 15 Ser Cys107 9 PRT Artificial Synthesized Peptide 107 Glu Val Asn Leu Asp Ala GluPhe Lys 1 5 108 6 PRT Artificial Synthesized peptide 108 Tyr Pro Tyr PheIle Pro 1 5 109 6 PRT Artificial Synthesized peptide 109 Tyr Pro Phe IlePro Leu 1 5

What is claimed is:
 1. An isolated peptide comprising an amino acidsequence comprising a Tyr-Pro-Tyr-Phe (SEQ ID NO:1) motif wherein thepeptide is capable of specifically binding to a BACE exosite.
 2. Thepeptide of claim 1 wherein one or more amino acid residues of theTyr-Pro-Tyr-Phe (SEQ ID NO:1) motif is replaced with a conservativeamino acid substitution.
 3. The peptide of claim 1 wherein the peptideconsists essentially of from about 5 to about 30 amino acids.
 4. Thepeptide of claim 1 wherein the amino acid sequence contains one or moremodified amino acid residues.
 5. The peptide of claim 4 wherein thepeptide contains a label selected from the group consisting of afluorescent label, a chromophore label, a radiolabel and a biotin label.6. The peptide of claim 1, wherein the peptide comprises a terminalmodification that enhances the resistance of the peptide to proteolysis.7. The peptide of claim 1 wherein the peptide comprises an amino acidsequence selected from the group consisting of Xaa-Tyr-Pro-Tyr-Phe (SEQID NO:3), Xaa-Tyr-Pro-Tyr-Phe-Xaa (SEQ ID NO:4),Xaa-Tyr-Pro-Tyr-Phe-Xaa-Xaa (SEQ ID NO:5), Tyr-Pro-Tyr-Phe-Xaa (SEQ IDNO:6) Tyr-Pro-Tyr-Phe-Xaa-Xaa (SEQ ID NO:7), His-Tyr-Pro-Tyr-Phe (SEQ IDNO:8), Tyr-Pro-Tyr-Phe-Ile (SEQ ID NO:2), Tyr-Pro-Tyr-Phe-Ile-Pro (SEQID NO:9), Tyr-Pro-Tyr-Phe-Ile-Pro-Leu (SEQ ID NO:10),Tyr-Pro-Tyr-Phe-Leu-Pro-Ile (SEQ ID NO:11), Tyr-Pro-Tyr-Phe-Xaa-Pro-Ile(SEQ ID NO:12), Tyr-Pro-Tyr-Phe-Xaa-Pro-Xaa (SEQ ID NO:13),His-Tyr-Pro-Tyr-Phe-Ile-Pro (SEQ ID NO:14) Tyr-Pro-Tyr-Phe-Leu (SEQ IDNO:15), Tyr-Pro-Tyr-Phe-Leu-Pro (SEQ ID NO:16),His-Tyr-Pro-Tyr-Phe-Leu-Pro (SEQ ID NO:17), andHis-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu (SEQ ID NO:18), wherein Xaa is anaturally or nonnaturally occurring amino acid residue.
 8. The peptideof claim 1 wherein the peptide comprises an amino acid sequence selectedfrom the group consisting of LTTYPYFIPLP (SEQ ID NO:44); TTYPYFIPLP (SEQID NO:45), TYPYFIPLP (SEQ ID NO:46), NLTTYPYFIPL (SEQ ID NO:48), YPYFIAL(SEQ ID NO:49), YPYFIPA (SEQ ID NO:50) YPYFIPB (SEQ ID NO:52), wherein Bindicates a benzophenone group, and HYPYFI (SEQ ID NO:54).
 9. Thepeptide of claim 1 comprising an amino acid sequence selected from thegroup consisting of Asn-Leu-Thr-Thr-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Pro (SEQID NO:19) and Ala-Leu-Tyr-Pro-Tyr-Phe-Leu-Pro-Ile-Ser-Ala-Lys (SEQ IDNO:20).
 10. The peptide of claim 1, wherein the peptide is cyclic.
 11. Acomposition comprising the peptide of claim 1 and a pharmaceuticallyacceptable carrier.
 12. An isolated peptide comprising an amino acidsequence having a WPXFI (SEQ ID NO:21) motif wherein the peptide iscapable of specifically binding to a BACE exosite.
 13. The peptide ofclaim 12 wherein one or more amino acid residues of the WPXFI (SEQ IDNO:21) motif is replaced with a conservative amino acid substitution.14. The peptide of claim 12 wherein the peptide consists essentially offrom about 5 to about 30 amino acids.
 15. The peptide of claim 12wherein the amino acid sequence contains one or more modified amino acidresidues.
 16. The peptide of claim 15 wherein the peptide contains alabel selected from the group consisting of a fluorescent label,chromophore label, a radiolabel and a biotin label.
 17. The peptide ofclaim 12, wherein the peptide comprises a terminal modification thatenhances the resistance of the peptide to proteolysis.
 18. The peptideof claim 12 comprising an amino acid sequence selected from the groupconsisting ofGlu-Thr-Trp-Pro-Arg-Phe-Ile-Pro-Tyr-His-Ala-Leu-Thr-Gln-Gln-Thr-Leu-Lys-His-Gln-Gln-His-Thr(SEQ ID NO:22);Thr-Ala-Glu-Tyr-Glu-Ser-Arg-Thr-Ala-Arg-Thr-Ala-Pro-Pro-Ala-Pro-Thr-Gln-His-Trp-Pro-Phe-Phe-Ile-Arg-Ser-Thr(SEQ ID NO:23) and His-Trp-Pro-Phe-Phe-Ile-Arg-Ser (SEQ ID NO:57).
 19. Acomposition comprising the peptide of claim 12 and a pharmaceuticallyacceptable carrier.
 20. An isolated peptide comprising the amino acidsequence YPBFIPL (SEQ ID NO:51), wherein the peptide is capable ofspecifically binding to a BACE exosite and wherein B indicates abenzophenone group.
 21. The peptide of claim 20 wherein one or moreamino acid residues of the YPBFIPL (SEQ ID NO:51) motif is replaced witha conservative amino acid substitution and wherein B indicates abenzophenone group.
 22. An isolated peptide comprising the amino acidsequence YPYFIP (SEQ ID NO:10), wherein the peptide is capable ofspecifically binding to a BACE exosite and wherein B indicates abenzophenone group.
 23. The peptide of claim 22 wherein one or moreamino acid residues of the YPYFIP (SEQ ID NO:10) motif is replaced witha conservative amino acid substitution and wherein B indicates abenzophenone group.
 24. The peptide of claim 20 wherein the peptideconsists essentially of from about 5 to about 30 amino acids.
 25. Thepeptide of claim 20 wherein the amino acid sequence contains one or moremodified amino acid residues.
 26. The peptide of claim 25 wherein thepeptide contains a label selected from the group consisting of afluorescent label, a chromophore label, a radiolabel and a biotin label.27. The peptide of claim 20, wherein the peptide comprises a terminalmodification that enhances the resistance of the peptide to proteolysis.28. A composition comprising the peptide of claim 20 and apharmaceutically acceptable carrier.
 29. An isolated nucleic acidencoding a BACE exosite binding peptide having an amino acid sequenceselected from the group consisting of Xaa-Tyr-Pro-Tyr-Phe (SEQ ID NO:3),Xaa-Tyr-Pro-Tyr-Phe-Xaa (SEQ ID NO:4), Xaa-Tyr-Pro-Tyr-Phe-Xaa-Xaa (SEQID NO:5), Tyr-Pro-Tyr-Phe-Xaa (SEQ ID NO:6) Tyr-Pro-Tyr-Phe-Xaa-Xaa (SEQID NO:7), His-Tyr-Pro-Tyr-Phe (SEQ ID NO:8), Tyr-Pro-Tyr-Phe-Ile (SEQ IDNO:2), Tyr-Pro-Tyr-Phe-Ile-Pro (SEQ ID NO:9),Tyr-Pro-Tyr-Phe-Ile-Pro-Leu (SEQ ID NO:10), Tyr-Pro-Tyr-Phe-Leu-Pro-Ile(SEQ ID NO:11), Tyr-Pro-Tyr-Phe-Xaa-Pro-Ile (SEQ ID NO:12),Tyr-Pro-Tyr-Phe-Xaa-Pro-Xaa (SEQ ID NO:13), His-Tyr-Pro-Tyr-Phe-Ile-Pro(SEQ ID NO:14) Tyr-Pro-Tyr-Phe-Leu (SEQ ID NO:15),Tyr-Pro-Tyr-Phe-Leu-Pro (SEQ ID NO:16), His-Tyr-Pro-Tyr-Phe-Leu-Pro (SEQID NO:17), and His-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu (SEQ ID NO:18) whereinXaa is a naturally or non-naturally occurring amino acid residue.
 30. Amethod of identifying a peptide that specifically binds to a BACEexosite comprising: (a) contacting BACE with at least one peptide; and(b) determining whether the peptide specifically binds to BACE at a siteother than the active site of BACE.
 31. A method of identifying amodulator of BACE activity comprising the steps of: (a) contacting acandidate modulator of BACE and a BACE exosite binding peptide in thepresence of BACE or a BACE variant including at least one BACE exosite;and (b) determining whether there is an increase or a decrease inbinding of the exosite binding peptide to BACE in the presence of thecandidate BACE modulator compared to binding of the exosite bindingpeptide to BACE in the absence of the candidate modulator.
 32. Themethod of claim 31 wherein BACE is an isolated BACE or an isolated BACEvariant.
 33. The method of claim 31 wherein BACE is a recombinant BACEor a recombinant BACE variant.
 34. The method of claim 31 wherein BACEis a BACE fusion protein.
 35. The method of claim 31 wherein the exositebinding peptide contains a label selected from the group consisting of afluorescent label, a chromophore label, a radiolabel and a biotin label.36. A compound identified by the method of claim
 31. 37. A compositioncomprising the compound of claim 36 and a pharmaceutically acceptablecarrier.
 38. A method of identifying a therapeutic for treating adisorder involving APP processing and beta-amyloid productioncomprising: (a) contacting BACE with a candidate BACE exosite bindingcompound; and (b) determining an amount of inhibition of APP processingand beta-amyloid production.
 39. A method of identifying a BACE exositebinding compound that inhibits beta-amyloid production comprising: (a)contacting a candidate exosite binding compound with a cell thatexpresses a beta-amyloid precursor protein and BACE, wherein the cell iscapable of secreting beta-amyloid protein in the absence of thecandidate exosite binding compound; and (b) determining whether thecandidate exosite binding compound reduces the amount of beta-amyloidprotein secreted by the cell in the absence of the candidate exositebinding compound.
 40. The method of claim 39 wherein the exosite bindingpeptide comprises a label selected from the group consisting of afluorescent label, a radiolabel and a biotin label.
 41. A method oftreating a neurological disorder comprising administering to a patientin need of such treatment a therapeutically effective amount of acompound, or a pharmaceutically acceptable salt or prodrug form thereof,wherein the compound specifically binds to a BACE exosite and modulatesBACE activity.